Alkylating agents

ABSTRACT

The present invention relates to a novel class of alkylating agents comprising a thieno-indole moiety linked to a DNA-binding moiety, which have cytotoxic activity and are useful in treating diseases such as cancer, cellular proliferation disorders and viral infections. The present invention also provides methods for preparing these compounds, pharmaceutical compositions comprising them and methods of treating diseases utilizing such compounds or the pharmaceutical composition containing them. The invention also relates to the use of this novel class of alkylating agents in the preparation of conjugates.

The present invention relates to a novel class of alkylating agentscomprising a thieno-indole moiety linked to a DNA-binding moiety,methods for their preparation, pharmaceutical composition containingthem and use thereof in treating certain mammalian tumors.

A wide range of chemicals is now available to treat cancers. Despite theefforts in anticancer research, cancer remains a looming and elusivetarget, therefore there is still a need for new anticancer agents.

Alkylating agents are cytotoxic agents that have been used for thetreatment of cancer for over six decades, yet their repertoire continuesto grow. These agents act during all phases of the cell cycle directlyon DNA, causing DNA strand breaks, leading to abnormal base pairing,inhibition of cell division and eventually resulting in cell death.

The present invention provides a novel class of alkylating agentscomprising a thieno-indole moiety linked to a DNA-binding moiety.

Thieno-indoles derivatives are described in GB2344818; some specificcompounds of the aforementioned patent applications are excluded fromthe present general formula.

Accordingly, a first object of the present invention is to provide acompound of formula (I) or (II)

wherein R1 and R2 taken together form a group (D) or (G):

wherein R5 is hydrogen, linear or branched C₁-C₄ alkyl, linear orbranched C₁-C₄ hydroxyalkyl or linear or branched C₁-C₄ aminoalkyl;R3 and R4 are, each independently, hydrogen or a group selected fromoptionally substituted linear or branched C₁-C₄ alkyl and linear orbranched C₁-C₄ hydroxyalkyl;R6 is a leaving group;T is null or N;BM is a DNA binding moiety of formula (V):

wherein:X is null, linear or branched C₁-C₄ alkyl, linear or branched C₂-C₄alkenyl or linear or branched C₂-C₄ alkynyl;Y and Y′ are independently an optionally substituted aryl or heteroaryl;U is a moiety of formula (VI) or (VII):

wherein R3 is as defined above;q is an integer from 0 to 4;L1 is hydrogen or L, wherein L is null or a conditionally-cleavablemoiety;W1 is null or a self-immolative system comprising one or moreself-immolative groups;Z1 is null or a peptidic, non peptidic or hydrid peptidic and nonpeptidic linker;provided that a compound of formula (I) or a compound of formula (II)wherein L1 is hydrogen is excluded when1) both T and X are null, q is 0 andY′ is an heterocyclyl moiety of formula (VIII), (VIII)′ or (VIII)″:

or2) both T and X are null, q is 1, U is a group of formula (VII),Y is an heterocyclyl moiety of formula (IX)

andY′ is an heterocyclyl moiety of formula (VIII)′″

wherein Q is N or O, and R21 is hydrogen or a group selected fromN(C₂H₅)₂ and C(NF)NH₂,andthe pharmaceutically acceptable salts thereof.

Cytotoxic drugs act on rapidly proliferating cells with differentmechanisms, usually by interfering directly or indirectly with DNAreplication. Although this therapy resulted effective in different tumortypes, it may suffer from some limitations: interfering with cellproliferation affects indeed also normal cells that proliferatefrequently, such as bone marrow, cells of the gastrointestinal tract andhair follicles. Drug concentrations that would completely eradicate thetumor may not be reached because of dose-limiting side effects on thesetissues leading immunosuppression, gastrointestinal tract toxicity andhair loss.

In addition cytotoxic drugs show in some cases non optimalphysicochemical properties and may lack of suitable pharmacokineticproperties limiting their use in patients.

Conjugation of cytotoxic drugs to molecules able to vehicle the drug andthus improving tumor targeting or able to modify its pharmacokineticproperties is one of the strategies that has been undertaken to solvethe above mentioned issues. Different examples of conjugation ofcytotoxic drugs with proteins, peptides or aptamer, polymers ornanoparticles allowing better target delivery, improving solubility andin some cases other pharmacokinetic properties such as increasing halflife or local concentration of the drug and improving drug performanceshave been reported. As a matter of facts, the resultant conjugates haveimproved characteristics in term of solubility, permeability into thecell, in vivo therapeutic window, controlled release, ability to reachthe target according to the nature of the specific molecule conjugatedwith the cytotoxic agent.

For this reason, there is an increasing demand for the development offunctionalized cytotoxic agents suitable to be conjugated with differenttypes of molecules.

The present invention also provides functionalized alkylating agentswhich, other than having cytotoxic activity, are also suitable to beconjugated with different types of nucleophiles.

Accordingly, a second object of the present invention is to provide acompound of formula (III) or (IV):

whereinBM is a DNA binding moiety of formula (V)′:

wherein:X, Y, U, Y′ and q are as defined above;A is an atom selected from —O—, —NH—, —CO—;A′ is null or A, wherein A is as defined above;L is null or a conditionally-cleavable moiety;W is null or a self-immolative system comprising one or moreself-immolative groups;Z is null or a peptidic, non peptidic or hybrid peptidic and nonpeptidic linker;RM is null or a reactive moiety attached to one or more of A, L, W or Zgroups;RM1 is null or a reactive moiety attached to one or more of L1, W1 or Z1groups;and R1, R2, R3, R4, R6, T, BM, L1, W1 and Z1 are as defined above;provided that1) a compound of formula (IV) is excluded when A′ is null and RM1 isnull;2) a compound of formula (III) or (IV) is excluded whena) both T and X are null, q is 0 andY′ is an heterocyclyl moiety of formula (VIII)^(IV)

whereinQ is —O—, —S—, —NR14, wherein R14 is hydrogen, C₁-C₄ alkyl or C₁-C₄hydroxyalkyl;

Q1 is —CH═ or —N═;

R7 and R8 are independently hydrogen, halogen, hydroxy, C₁-C₄ alkoxy,cyano, —NCOOR3, —C(NH)—NH₂ or NR3R4,wherein R3 and R4 are as defined above;orb) both T and X are null, q is 1 or 2, U is a group of formula (VII),Y is a heterocyclyl moiety of formula (IX)′:

and Y′ is a heterocyclyl moiety of formula (VIII)^(IV)

wherein Q, Q1, R7 and R8 are as defined above;and the pharmaceutically acceptable salts thereof.

It is noted that when L1 is hydrogen or a conditionally-cleavablemoiety, and the O-L1 bond is broken so generating a OH function, thenthe compounds of formula (II) or (IV) may be transformed in a compoundof formula (I) or (III) respectively, through the well reviewed reactionmechanism reported in the literature (see e.g. Baiard, R; et al., J. Am.Chem. Soc. 1963, 85, 567-578; Zhao, R. Y. et al. J. Med. Chem. 2012, 55,766-782.)

In addition, it is to be noted that a compound of formula (III) has onefunctionalization

while a compound of formula (IV) may have one or twofunctionalization(s).

Specifically, a compound of formula (IV) has functionalization when:

A′ is A and L1 is hydrogen, as in a compound of formula (IV)′

orA′ is null and L1 is not hydrogen, as in a compound of formula (IV)′″,

since L, W, Z and/or RM cannot be attached directly to BM.A compound of formula (IV) has two functionalizations whenA′ is A and L1 is L, as in a compound of formula (IV)″

The present invention also provides methods of synthesizing thecompounds of formula (I), (II), (III) and (IV), prepared through aprocess consisting of standard synthetic transformations and isomers,tautomers, hydrates, solvates, complexes, metabolites, prodrugs,carriers, N-oxides.

The present invention also provides a method for treating cancer, whichcomprises administering to a mammal, in need thereof, an effectiveamount of a compound of formula (I), (II), (III) or (IV) as definedabove. The mammal in need thereof may be for example a human.

The present invention also provides a compound of formula (I), (II),(III) or (IV), as defined above, for use in a method of treating cancer,cellular proliferation disorders and viral infections.

Preferably, a compound of formula (I), (II), (III) or (IV), as definedabove is for use in a method of treating specific types of cancers,including but not limited to: carcinoma such as bladder, breast, colon,kidney, liver, lung, including small cell lung cancer, esophagus,gall-bladder, ovary, pancreas, stomach, cervix, thyroid, prostate, andskin, including squamous cell carcinoma; hematopoietic tumors oflymphoid lineage including leukemia, acute lymphocitic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell-lymphoma, Hodgkin'slymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma and Burkitt'slymphoma; hematopoietic tumors of myeloid lineage, including acute andchronic myelogenous leukaemias, myelodysplastic syndrome andpromyelocytic leukemia; tumors of mesenchymal origin, includingfibrosarcoma and rhabdomyosarcoma; tumors of the central and peripheralnervous system, including astrocytoma neuroblastoma, glioma andschwannomas; other tumors, including melanoma, seminoma,teratocarcinoma, osteosarcoma, xeroderma pigmentosum, keratoxanthoma,thyroid follicular cancer, Kaposi's sarcoma and mesothelioma.

Furthermore, a compound of formula (I), (II), (III) or (IV), as definedabove is for use in a method of treating specific cellular proliferationdisorders such as, for example, benign prostate hyperplasia, familialadenomatosis polyposis (FAP), neurofibromatosis, psoriasis, vascularsmooth cell proliferation associated with atherosclerosis, pulmonaryfibrosis, arthritis, glomerulonephritis and post-surgical stenosis andrestenosis.

In addition, a compound of formula (I), (II), (III) or (IV), as definedabove is for use in a method of inhibiting tumor angiogenesis andmetastasis, as well as in a method of treating organ transplantrejection and host versus graft disease.

The present invention also provides a pharmaceutical compositioncomprising a therapeutically effective amount of compounds of formula(I), (II), (III) or (IV) or a pharmaceutically acceptable salt thereofas defined above and at least one pharmaceutically acceptable excipient,carrier and/or diluent.

The present invention further provides a pharmaceutical compositioncomprising a compound of formula (I), (II), (III) or (IV) and one ormore chemotherapeutic agents.

The present invention further provides a pharmaceutical compositioncomprising a compound of formula (I), (II), (III) or (IV) in combinationwith known anticancer treatments such as radiation therapy orchemotherapy regimen in combination with cytostatic or cytotoxic agents,antibiotic-type agents, alkylating agents, antimetabolite agents,hormonal agents, immunological agents, interferon-type agents,cyclooxygenase inhibitors (e.g. COX-2 inhibitors), matrixmetalloproteaseinhibitors, telomerase inhibitors, tyrosine kinase inhibitors,anti-growth factor receptor agents, anti-HER2 agents, anti-EGFR agents,anti-angiogenesis agents (e.g. angiogenesis inhibitors), farnesyltransferase inhibitors, ras-raf signal transduction pathway inhibitors,cell cycle inhibitors, other cdks inhibitors, tubulin binding agents,topoisomerase I inhibitors, topoisomerase II inhibitors, and the like.

Additionally, the invention provides a product comprising a compound offormula (I), (II), (III), or (IV) or a pharmaceutically acceptable saltthereof, as defined above, and one or more chemotherapeutic agents, as acombined preparation for simultaneous, separate or sequential use inanticancer therapy.

In yet another aspect the invention provides a compound of formula (I),(II), (III) or (IV) or a pharmaceutically acceptable salt thereof, asdefined above, for use as a medicament.

Moreover the invention provides the use of a compound of formula (I),(II), (III) or (IV) or a pharmaceutically acceptable salt thereof, asdefined above, in the manufacture of a medicament with anticanceractivity.

Finally, the invention provides the use of a compound of formula (I),(II), (III) or (IV) or a pharmaceutically acceptable salt thereof, asdefined above, in the preparation of conjugates.

Unless stated otherwise, the following terms and phrases as used hereinare intended to have the following meanings.

With the term “linear or branched C₁-C₄ alkyl” we intend any of thegroups such as, for instance, methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl.

With the term “linear or branched C₁-C₄ hydroxyalkyl” we intend any ofthe groups such as, for instance, 2-hydroxyethyl, 3-hydroxypropyl,2-hydroxypropyl, 4-hydroxybutyl, 3-hydroxybutyl, 2-hydroxybutyl.

With the term “linear or branched C₁-C₄ alkoxy”, we intend any of thegroups such as, for instance, methoxy, ethoxy, propoxy, etc.

With the term “halogen” we intend a fluorine, chlorine, bromine oriodine.

With the term “linear or branched C₁-C₄ aminoalkyl” we intend any of thegroups such as, for instance, 2-aminoethyl, 3-aminopropyl, 4-aminobutyl,3-aminobutyl, etc.

The term “C₃-C₈ cycloalkyl” as used herein refers to a saturated orunsaturated non-aromatic all-carbon monocyclic ring, which may consistof one ring or two or more rings fused together. Examples include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl,cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl,decalinyl, and 1,4-cyclohexadienyl.

The term “heterocyclyl” as used herein refers to a saturated orunsaturated non-aromatic C₄-C₈ carbocyclic ring which may consist of onering or two or more rings fused together, wherein from 1 to 4 carbonatoms are replaced by heteroatoms such as nitrogen, oxygen, sulfur,wherein said heteroatoms may be directly connected to each other;nitrogen and sulfur may optionally be oxidized and nitrogen mayoptionally be quaternized. Non limiting examples of heterocyclyl groupsare, for instance, tetrahydrofuranyl, pyrrolidinyl, piperidinyl,1,4-dioxanyl, decahydroquinolinyl, piperazinyl, oxazolidinyl andmorpholinyl.

The term “aryl” as used herein refers to a mono-, bi- orpoly-carbocyclic hydrocarbon from 1 to 4 ring systems, optionallyfurther fused or linked to each other by single bonds, wherein at leastone of the carbocyclic rings is aromatic, wherein the term “aromatic”refers to completely conjugated π-electron bond system. Non limitingexamples of such aryl groups are phenyl, α- or β-naphthyl or anthracenylgroups.

The term “heteroaryl” as used herein refers to aromatic heterocyclicrings, typically 4- to 7-membered heterocycles, with from 1 to 4heteroatoms selected among oxygen, nitrogen and sulfur, wherein nitrogenand sulfur may optionally be oxidized and nitrogen may optionally bequaternized; said heteroaryl ring can be optionally further fused orlinked to one or two or more rings fused together, aromatic andnon-aromatic carbocyclic and heterocyclic rings. Heteroatoms may bedirectly connected to each other. Examples of heteroaryl groups include,but are not limited to, pyridinyl, pyrimidyl, furanyl, pyrrolyl,triazolyl, pyrazolyl, pyrazinyl, oxazolyl, isoxazolyl, thiazolyl,imidazolyl, thienyl, indolyl, benzofuranyl, benzimidazolyl,benzothiazolyl, purinyl, indazolyl, benzotriazolyl, benzisoxazolyl,quinoxalinyl, isoquinolyl, and quinolyl. In one embodiment, a heteroarylgroup comprises from 1 to 4 heteroatoms. It should be noted that “C₁heteroaryl group” denotes that there is only one carbon present in thering system of the heteroaromatic group (carbon atoms in optionalsubstituents are thus not counted). An example of such a heteroaromaticgroup is a tetrazolyl group.

The term “leaving group” refers to a group that can be substituted byanother group in a substitution reaction. Such leaving groups arewell-known in the art and examples include, but are not limited to, anhalide (fluoride, chloride, bromide, and iodide), an azide, a sulfonate(e.g., an optionally substituted C₁-C₆ alkanesulfonate, such asmethanesulfonate and trifluoromethanesulfonate, or an optionallysubstituted C₇-C₁₂ alkylbenzenesulfonate, such as p-toluenesulfonate),succinimide-N-oxide, p-nitrophenoxide, pentafluorophenoxide,tetrafluorophenoxide, a carboxylate, an aminocarboxylate (carbamate) andan alkoxycarboxylate (carbonate). For substitutions at saturated carbon,halides and sulfonates are preferred leaving groups. For substitutionsat a carbonyl carbon a halide, succinimide-N-oxide, p-nitrophenoxide,pentafluorophenoxide, tetrafluorophenoxide, a carboxylate, or analkoxycarboxylate (carbonate) may for example be used as a leavinggroup. The term “leaving group” also refers to a group that iseliminated as a consequence of an elimination reaction, e.g., anelectronic cascade reaction or a spirocyclization reaction. In thisinstance, an halide, a sulfonate, an azide, an aminocarboxylate(carbamate) or an alkoxycarboxylate (carbonate) may for example be usedas a leaving group.

The term “active ester” refers to a functional group in which the alkoxygroup of the ester moiety is a good leaving group. Examples of suchalkoxy groups include, but are not limited to, succinimide-N-oxide,p-nitrophenoxide, pentafluorophenoxide, tetrafluorophenoxide,1-hydroxybenzotriazole, and 1-hydroxy-7-azabenzotriazole, and groupswith comparable leaving capability. Unsubstituted alkyl-based alkoxygroups such as methoxy, ethoxy, isopropoxy, and t-butoxy do not qualifyas good leaving groups and methyl, ethyl, isopropyl, and t-butyl estersare therefore not considered to be active esters.

The term “nucleophiles” refers to molecules that bear a nucleophilicgroup. The term “nucleophilic group” refers to a species that donates anelectron-pair to an electrophilic group to form a chemical bond in achemical reaction. Examples of such nucleophilic groups include, but arenot limited to halogens, amines, nitrites, azides, alcohols, alkoxydeanions, carboxylate anions, thiols, thiolates, etc.

The term “electrophilic group” refers to a species that accepts anelectron-pair from a nucleophilic group to form a chemical bond in achemical reaction. Examples of such electrophilic groups include, butare not limited to esters, aldehydes, amides, ketons, etc.

The term “alkylating moiety” refers to the structure that remain afterbreaking of one or more cleavable bonds and that may or may not becovalently bound to the nucleic acid strand.

The term “unnatural amino acid” refers to the D-stereoisomer of thenaturally occurring amino acid.

Pharmaceutically acceptable salts of the compounds of formula (I), (II),(III) or (IV) include the acid addition salts with inorganic or organicacids, e.g., nitric, hydrochloric, hydrobromic, sulfuric, perchloric,phosphoric, acetic, trifluoroacetic, propionic, glycolic, fumaric,lactic, oxalic, malonic, malic, maleic, tartaric, citric, benzoic,cinnamic, mandelic, methanesulphonic, isethionic and salicylic acid.

Pharmaceutically acceptable salts of the compounds of formula (I), (II),(III) or (IV) also include the salts with inorganic or organic bases,e.g., alkali or alkaline-earth metals, especially sodium, potassium,calcium ammonium or magnesium hydroxides, carbonates or bicarbonates,acyclic or cyclic amines.

If a stereogenic center or another form of an isomeric center is presentin a compound of the present invention, all forms of such isomer orisomers, including enantiomers and diastereomers, are intended to becovered herein. Compounds containing a stereogenic center may be used asa racemic mixture, an enantiomerically enriched mixture, or the racemicmixture may be separated using well-known techniques and an individualenantiomer may be used alone. In cases in which compounds haveunsaturated carbon-carbon double bonds, both the cis (Z) and trans (E)isomers are within the scope of this invention.

In cases when compounds can exist in tautomeric forms, each form iscontemplated as being included within this invention whether existing inequilibrium or predominantly in one form.

The DNA-Binding Moiety BM

The BM moiety is a binding moiety that binds or associates the compoundof formula (I), (II), (III) or (IV) with the double strand of the DNA.The binding moiety can improve affinity properties of the derivatives tothe DNA or improve alkylating reactivity of the alkylating agent ortarget different sequences of the DNA so to modulate target specificityof the compounds.

Preferably, in a compound of formula (I) or (II) the BM moiety is agroup of formula (V) as defined above,

whereinX is null or a C₂-C₄ alkenyl of formula (XIV):

wherein R3, independently the same or different, is as define above;U and q are as defined above;Y, if present, is a group selected from:

andY′ is a group selected from:

wherein R3 is as defined above, and R15, R16 and R20 are independentlyhydrogen, halogen, hydroxy, NO₂, an optionally substituted linear orbranched C₁-C₆ alkyl or C₁-C₆ alkoxy, cyano, —COOH, —CONH—R3,—N—C(O)O—R3, —C(NH)—NH₂ or NR3R4 in which R3 and R4 are as definedabove.

According to the present invention and unless otherwise provided, theabove R3, R15, R16 and R20 groups may be optionally substituted, in anyof their free positions, by one or more groups, for instance 1 to 3groups, independently selected from: alkoxy, hydroxy, amino, linear orbranched C₁-C₄ alkylamino, dialkylamino, aminocarbonyl, heterocyclyl andheterocyclylalkyl. In their turn, whenever appropriate, each of theabove substituent may be further substituted by one or more of theaforementioned groups.

More preferably, in a compound of formula (I) or (II) the BM moiety is agroup of formula (V) as defined above, wherein X and U are as definedabove, q is an integer from 0 to 2;

Y, if present, is a group selected from

wherein R3 is as defined above;andY′ is a group selected from:

wherein R3 is as defined above, and R15, R16 and R20 are independentlyhydrogen or methoxy.

Preferably, in a compound of formula (III) or (IV), the BM moiety is agroup of formula (V)′ as defined above, wherein

X is null or a C₂-C₄ alkenyl of formula (XIV) as defined above;U and q are as defined above;andY, if present, and Y′ are independently selected from:

wherein R3, R15, R16 and R20 are as defined above.

More preferably, in a compound of formula (III) or (IV), the BM moietyis a group of formula (V)′ as defined above, wherein X and U are asdefined above, q is an integer from 0 to 2;

Y, if present, is selected from:

wherein R3 is as defined above; andY′ is a group selected from:

wherein R3 is as defined above and R15, R16 and R20 are independentlyhydrogen or methoxy.

The Conditionally-Cleavable Moiety L or L1

The L or L1 moiety, if present, is a conditionally-cleavable group thatcan be cleaved by a chemical, photochemical, physical biological orenzymatic process upon being brought in or under certain conditions. Oneof these condition may for example be bringing a compound of theinvention in an aqueous environment, which leads to hydrolysis of Land/or L1, or bringing a compound of the invention in an environmentthat contains an enzyme that recognizes and cleaves L and/or L1, orbringing a compound of the invention under reducing conditions, whichleads to reduction and/or removal of L and/or L1, or bringing a compoundof the invention under oxidizing conditions, which leads to oxidationand removal of L and/or L1, or bringing a compound of the invention incontact with radiation, e.g., UV light, which leads to cleavage, orbringing a compound of the invention in contact with heat, which leadscleavage of L and/or L1. This condition may be met directly afteradministrating a compound of this invention to an animal, e.g., amammal, for example a human, due to the presence of ubiquitous enzymesin the circulation. Alternatively, said condition may be met when thecompound localizes to a specific organ, tissue, cell, subcellulartarget, or bacterial, viral, or microbial target, for example by thepresence of internal factors (e.g., target-specific enzymes or hypoxia)or application of external factors (e.g., radiation, magnetic fields).

Cleavage of L or L1 means that the bond between A and L in a compound offormula (III) or (IV), or between the oxygen and L1 in a compound offormula (II) or (IV) is broken:

It is noted that in a compound of formula (IV), twoconditionally-cleavable groups can be present. In this case the twomoieties may or may not be the same and may or may not require the sameconditions for cleavage.

In one embodiment, L and/or L1 can be moieties that are cleaved by anenzyme or hydrolytic conditions present in the vicinity or inside thetarget cells as compared to other parts of the body, or by an enzyme orhydrolytic conditions, present only in the vicinity of or inside thetarget cells. It is important to recognize that if target sitespecificity is achieved solely based upon the selective transformationand/or cleavage of said L at the target site, the condition causing thecleavage should preferably, at least to a certain degree, be targetsite-specific. In one embodiment, cleavage of L occurs intracellularly.

In another embodiment, cleavage of L occurs extracellularly.

In another embodiment, cleavage of L and/or L1 can occur by a ubiquitousintracellular enzyme.

In one preferred embodiment L and/or L1 may be a moiety that can becleaved by ubiquitous enzymes, e.g., esterases that are present in thecirculation or intracellular enzymes, such as for example proteases andphosphatases, or by pH-controlled hydrolysis. L and/or L1 may thereforeform, optionally together with the connecting atom(s) A or oxygen, acarbonate, carbamate, urea, ester, amide, imine, disulfide, ether,acetal, ketal or phosphate group that can be cleaved in vivo.

In a more preferred embodiment A is —O—, and L and L1 are independentlynull or a group selected from: —NHCO—R9 (Xa); —NHCONH—R9 (Xb); —NHCOO—R9(Xc); —NH—R9 (Xd);

wherein:R9 and R10 are, each independently, null, hydrogen, hydroxy or anoptionally substituted group selected from linear or branched C₁-C₄alkyl, linear or branched C₁-C₄ hydroxyalkyl, linear or branched C₁-C₄sulfhydrylalkyl and linear or branched C₁-C₄ aminoalkyl;n is an integer from 0 to 2 andn1 is an integer from 0 to 4.

According to the present invention and unless otherwise provided, theabove R9 and R10 groups may be optionally substituted, in any of theirfree positions, by one or more groups, for instance 1 to 3 groups,independently selected from: halogen, linear or branched C₁-C₄ alkyl,polyfluorinated alkyl, linear or branched C₁-C₄ alkoxy, polyfluorinatedalkoxy, hydroxy, amino, linear or branched C₁-C₄ alkylamino,dialkylamino, C₁-C₄ alkylcarbonyl, C₃-C₈ cycloalkyl, cycloalkylalkyl,heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl andheteroarylalkyl.

In another more preferred embodiment A is —N—, and L and L1 areindependently null or a group selected from:

wherein:R9 and n1 are as defined above.

In another more preferred embodiment A is —CO—, and L and L1 areindependently null or a group selected from:

wherein:R9, R10, and n1 are as defined above.

The Self-Immolative System W or W1

The W or W1 group, if present, is a self-immolative system that in acompound of formula (III) and (IV) tethers in a stable way from one sidea moiety L or A (if L is null), to a moiety Z or RM (if Z is null); in acompound of formula (II) tethers in a stable way from one side a moietyL1 or oxygen (if L1 is null), to Z1 or additionally to RM1 (if Z1 isnull) in compound of formula (IV). The L-W, A-W, L1-W1 or O—W1 bond canbecome labile upon activation by a chemical, photochemical, physical,biological or enzymatic process upon being brought in or under certaincondition, as described above, leading optionally to the release of thecorresponding moieties:

It is noted that in compound of formula (IV), two self-immolativesystems can be present. In this case the two systems may or may not bethe same and may or may not require the same conditions for cleavage.

A self-immolative system may be incorporated in a compound of formula(II), (III) or (IV) for example to improve solubility or to improvespace between the alkylating moiety and the reactive moiety; in additionsaid self-immolative system can modulate the reactivity of RM or RM1versus nucleophfiles.

Self-immolative systems are known to the person skilled in the art, seefor example those described in WO2002/083180 and WO2004/043493; or thosedescribed in Tranoy-Opalinsi, A. et al., Anticancer Agents in MedicinalChemistry, 2008, 8, 618-637. Other examples of self-immolative systemsinclude, but are not limited to, optionally substituted 4-aminobutyricacid amides, appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2]ring systems or 2-aminophenylpropionic acid amides [see WO 2005/079398,WO 2005/105154 and WO 2006/012527; Greenwald, R. B., et al., Adv. DrugDelivery Rev. 2003, 55, 217-250; Kingsbury, W. D.; et al., J. Med. Chem.1984, 27, 1447-1451].

In one preferred embodiment W or W1 may form together with theconnecting atom(s) L, L1, A, Z, Z1, RM, RM1 or oxygen, a carbonate,carbamate, urea, ester, amide, ether or thioamide linkage group that canbe optionally cleaved upon activation.

In preferred embodiment, W and W1 are independently null or aself-immolative system, comprising one or more self-immolative groupsindependently selected from:

whereinone of R9 and R10 is null and the other is as defined above;R11 and R12 are, each independently, hydrogen, halogen, methyl, ethyl orlinear or branched C₁-C₄ hydroxymethyl;m is an integer from 0 to 3; andA₁ is CH₂, CH₂N—R12 or N—R12, wherein R12 is as defined above.

In another more preferred embodiment, W and W1 are independently null ora group selected from:

wherein one of R9 and R10 is null and the other is as defined above; andR11 is as defined above.

The Z or Z1 Linker

The Z or Z1 linker, if present, can be peptidic (Z_(a)), non-peptidic(Z_(b)) or hybrid, wherein said hybrid linker is peptidic andnon-peptidic (Z_(c)); in a compound of formula (II), (III) or (IV) saidZ or Z1 linker can be cleaved from W or W1, respectively, by a chemical,photochemical, physical biological or enzymatic process upon beingbrought in or under certain conditions, as described above:

The Z or Z1 linker may be linear or branched.

The linkage between Z or Z1 and its left hand side moiety or between Zor Z1 and, optionally, RM or RM1, may be an amide, a carbonate, adisulfide or a carbamate linkage.

In one embodiment both of Z and Z1 are null; in another embodiment oneof Z or Z1 is null.

In another embodiment Z, Z1 is a peptidic linker Z_(a) that can becleaved by a proteolytic enzyme, plasmin, a cathepsin, β-glucuronidase,a galactosidase, prostate-specific antigen (PSA), urokinase-typeplasminogen activator (u-PA) or a member of the family of matrixmetalloproteinases.

In another embodiment Z, Z1 is a non-peptidic linker Z_(b) that maycontain one or more non-peptidic water-soluble moieties. In this casethe linker contributes to the water solubility of a compound of formula(II), (III) or (IV).

In another embodiment Z_(b) is a non-peptidic linker that may containone or more non-peptidic moieties that reduce(s) aggregation of acompound of formula (II), (III) or (IV), which may or may not be amoiety/moieties that also increase(s) the water solubility of a compoundof formula (II), (III) or (IV).

For example, non-peptidic water-soluble Z_(b) linkers may contain anoligoethylene glycol or polyethylene glycol moiety or a derivativethereof.

In another embodiment Z, Z1 is a hybrid linker Z_(c) that can containsboth, peptidic and non peptidic residues of general formula

Z_(a)—Z_(b)

wherein Z_(a) and Z_(b) are independently a peptidic linker or anon-peptidic linker. Hybrid linkers may contribute to solubility ofcompound of formula (II), (III) or (IV) and/or be a substrate that canbe cleaved by proteolytic enzyme, for example by a member of the familyof matrix metalloproteinases.

In a preferred embodiment, Z_(a) is a single amino acid, a dipeptide, atripeptide, a tetrapeptide, or an oligopeptide moiety comprising naturalL-amino acids, unnatural D-amino acids, synthetic amino acids, or anycombination thereof, wherein one of the C-terminal or the N-terminalamino acid residue is linked to W, L or A or to W1, L1, or oxygen and,the other terminal amino acid ends with a COOH or NH2 group or isoptionally linked to RM or RM1.

In a more preferred embodiment Z_(a) is a dipeptide or a tripeptide,linked via its C-terminus to W or W1, or to L when W is null, or to L1when W1 is null, or to A when W and L are both null, or to 0 when W1 andL1 are both null.

In another more preferred embodiment, the C-terminal amino acid residueof the dipeptide or of the tripeptide is selected from glycine, alanine,arginine and citrulline; and the N-terminal amino acid residue isselected from any natural or unnatural amino acid; preferably, in caseof the tripeptide, the middle amino acid residue is selected fromalanine, valine, leucine, isoleucine, methionine, phenylalanine andproline.

In another more preferred embodiment Z_(a) comprises a pentapeptide,wherein the C-terminal amino acid is selected from any natural orunnatural amino acid and the N-terminal amino acid residue is6-aminohexanoic acid.

In a preferred embodiment Z_(b) may contain an oligoethylene glycol orpolyethylene glycol moiety or a derivative thereof.

In a more preferred embodiment Z_(b) is a group selected from:

whereinone of R9 and R10 is null and the other is are as defined above; andp is an integer from 1 to 20;

In a preferred embodiment Z_(c) is a hybrid moiety comprising

a peptidic moiety Z_(a), wherein Z_(a) is a single amino acid, atripeptide or a tetrapeptide, comprising natural L amino acids andunnatural D amino acids; anda non-peptidic moiety Z_(b) comprising an oligoethylene glycol orpolyethylene glycol moiety or a derivative thereof.

The Reactive Moiety RM or RM1

The RM or RM1 moiety, if present, is an electrophilic group that canreact with nucleophiles, i.e. molecules that bear a nucleophilic group,under relatively mild conditions and without the need of priorfunctionalization of the reactive moiety, said reaction between thereactive moiety and said nucleophile will only require the applicationof some heat, pressure, a catalyst, acid, and/or base.

Therefore, when one of RM or RM1 moiety is present, a compound offormula (III) o (IV) conjugates with different types of nucleophiles.

When both RM and RM1 moiety are null, a compound of formula (III) or(IV) conjugates with different types of electrophiles, i.e. moleculesthat bear an electrophilic group, through one or more of thenucleophilic groups that are present on the A, L, L1, W, W1, Z and Z1moiety(ies).

In a compound of formula (III) the RM moiety can be connected to one ormore of the A, L, W or Z groups; in a compound of formula (IV), RM canbe connected either to one or more of the A, L, W or Z groups and/or toone ore more of the L1, W1, Z1 groups or to the oxygen atom:

Examples of reactive moieties include, but are not limited to, carbamoylhalide, acyl halide, active ester, anhydride, α-haloacetyl,α-haloacetamide, maleimide, isocyanate, isothiocyanate, disulfide,thiol, hydrazine, hydrazide, sulfonyl chloride, aldehyde, methyl ketone,vinyl sulfone, halomethyl, and methyl sulfonate.

In one preferred embodiment of the invention, when the nucleophilicgroup of the nucleophile is NH, NH₂ or OH, RM and RM1 are independentlynull or a group selected from

wherein R13 is C₁-C₃ alkyl or an electron-withdrawing group comprisingNO₂ and CN group;r is an integer from 0 to 7; andR11 and R12 are as defined above.

In another preferred embodiment of the invention, when the nucleophilicgroup of the nucleophile is COOH, RM and RM1 are independently null or agroup selected from

In another preferred embodiment of the invention, when the nucleophilicgroup of the nucleophile is SH, RM and RM1 are independently null or agroup selected from

wherein R13 is as defined above.

In a most preferred embodiment at least one of RM and RM1 is not null.

Preferably, the present invention provides compounds of formula (I) or(II) as defined above, characterized in that R1 and R2 taken togetherform a group (D), wherein R5 is linear or branched C₁-C₄ alkyl.

More preferably, the present invention provides compounds of formula(II) as defined above, characterized in that R6 is halide and

L1 is hydrogen or a conditionally-cleavable moiety of formula (Xj)

wherein R9 is hydrogen, hydroxy or an optionally substituted groupselected from linear or branched C₁-C₄ alkyl, linear or branched C₁-C₄hydroxyalkyl, linear or branched C₁-C₄ sulfhydrylalkyl and linear orbranched C₁-C₄ aminoalkyl.

Preferably, the present invention provides compounds of formula (III) or(IV) as defined above, characterized in that R1 and R2 taken togetherform a group (D), wherein R5 is linear or branched C₁-C₄ alky.

More preferably, the present invention provides compounds of formula(IV) as defined above, characterized in that R6 is halide; and

A′ is null and L1 is L, wherein L is as defined above.

More preferably, the present invention provides compounds of formula(III) or (IV) as defined above, characterized in that L and L1 areindependently null or a conditionally-cleavable moiety selected fromNHCO—R9 (Xa); —NHCONH—R9 (Xb); —NHCOO—R9 (Xc); —NH—R9 (Xd);

wherein:R9 and R10 are, each independently, null, hydrogen, hydroxy or anoptionally substituted group selected from linear or branched C₁-C₄alkyl, linear or branched C₁-C₄ hydroxyalkyl, linear or branched C₁-C₄sulfhydrylalkyl and linear or branched C₁-C₄ aminoalkyl;n is an integer from 0 to 2 andn1 is an integer from 0 to 4.

More preferably, the present invention provides compounds of formula(III) or (IV) as defined above, characterized in that W and W1 areindependently a self-immolative system comprising one or moreself-immolative groups independently selected from:

whereinone of R9 and R10 is null and the other is as defined above;R11 and R12 are, each independently, hydrogen, halogen, methyl, ethyl orlinear or branched C₁-C₄ hydroxymethyl;m is an integer from 0 to 3; andA₁ is CH₂, CH₂N—R12 or N—R12, wherein R12 is as defined above.

More preferably, the present invention provides compounds of formula(III) or (IV) as defined above, characterized in that Z and Z1 areindependently a peptidic linker, preferably a dipeptide or a tripeptidelinker, or is a non peptidic linker containing an oligoethylene glycolor polyethylene glycol moiety or a derivative thereof selected from:

whereinone of R9 and R10 is null and the other is are as defined above; andp is an integer from 1 to 20.

More preferably, the present invention provides compounds of formula(III) or (IV) as defined above, characterized in that RM and RM1 areindependently a reactive moiety selected from

wherein R13 is C₁-C₃ alkyl or an electron-withdrawing group comprisingNO₂ and CN group;r is an integer from 0 to 7; andR11 and R12 are as defined above.

Specific, not limiting, preferred compounds (compd.) of the presentinvention, optionally in the form of a pharmaceutically acceptable salt,are the following:

-   1)    N-(6-{[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1H-indol-3-yl)-1H-indole-6-carboxamide,-   2)    N-(5-{[(8R)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1-methyl-1H-pyrrol-3-yl)-1-methyl-4-{[(1-methyl-4-nitro-1H-pyrrol-2-yl)carbonyl]amino}-1H-pyrrole-2-carboxamide,-   3)    (2E)-1-[(8R)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)prop-2-en-1-one,-   4)    (2E)-1-[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)prop-2-en-1-one,-   5)    N-(6-{[(3bR,4aS)-3-methyl-8-oxo-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-3-yl)-1H-indole-6-carboxamide,-   6)    1-methyl-N-(1-methyl-5-{[(3bS,4aR)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-pyrrol-3-yl)-4-{[(1-methyl-4-nitro-1H-pyrrol-2-yl)carbonyl]amino}-1H-pyrrole-2-carboxamide,-   7)    (3bS,4aR)-3-methyl-6-[(2E)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)prop-2-enoyl]-4,4-a,5,6-tetrahydro-8H-cyclopropa[c]thieno[3,2-e]indol-8-one,-   8)    (3bR,4aS)-3-methyl-6-[(2E)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)prop-2-enoyl]-4,4-a,5,6-tetrahydro-8H-cyclopropa[c]thieno[3,2-e]indol-8-one,-   9)    (3bS,4aR)—N-(5-{[5-({5-[(3-amino-3-oxopropyl)carbamoyl]-1-methyl-1H-pyrrol-3-yl}carbamoyl)-1-methyl-1H-pyrrol-3-yl]carbamoyl}-1-methyl-1H-pyrrol-3-yl)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indole-6(8H)-carboxamide,-   10)    (8R)—N-(5-{[5-({5-[(3-amino-3-oxopropyl)carbamoyl]-1-methyl-1H-pyrrol-3-yl}carbamoyl)-1-methyl-1H-pyrrol-3-yl]carbamoyl}-1-methyl-1H-pyrrol-3-yl)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6-carboxamide,-   11)    (8S)—N-(5-{[5-({5-[(3-amino-3-oxopropyl)carbamoyl]-1-methyl-1H-pyrrol-3-yl}carbamoyl)-1-methyl-1H-pyrrol-3-yl]carbamoyl}-1-methyl-1H-pyrrol-3-yl)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6-carboxamide,-   12)    (3bR,4aS)—N-(5-{[5-({5-[(3-amino-3-oxopropyl)carbamoyl]-1-methyl-1H-pyrrol-3-yl}carbamoyl)-1-methyl-1H-pyrrol-3-yl]carbamoyl}-1-methyl-1H-pyrrol-3-yl)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indole-6(8H)-carboxamide,-   13)    N-(3-{(1E)-3-[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]-3-oxoprop-1-en-1-yl}-1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-indole-2-carboxamide,-   14)    N-(3-{(1E)-3-[(8R)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]-3-oxoprop-1-en-1-yl}-1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-indole-2-carboxamide,-   15)    N-(3-{(1E)-3-[(3bR,4aS)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-y]-3-oxoprop-1-en-1-yl}-1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-indole-2-carboxamide,-   16)    N-(3-{(1E)-3-[(3bR,4aS)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-y]-3-oxoprop-1-en-1-yl}-1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-indole-2-carboxamide,-   17)    N-(2-{[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1H-indol-5-yl)-5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indole-2-carboxamide,-   18) tert-butyl    {2-[(2-{[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1H-indol-5-yl)carbamoyl]-1H-indol-5-yl}carbamate,-   19)    5-amino-N-(2-{[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-5a,7,8,8a-tetrahydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1H-indol-5-yl)-1H-indole-2-carboxamide,-   20) tert-butyl    {2-[(2-{[(3bR,4aS)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-5-yl)carbamoyl]-1H-indol-5-yl}carbamate,-   21)    5-amino-N-(2-{[(3bR,4aS)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-5-yl)-1H-indole-2-carboxamide,-   22)    (8S)-6-({5-[({5-[(tert-butoxycarbonyl)amino]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl    4-methylpiperazine-1-carboxylate,-   23)    (8S)-6-[(5-{[(5-amino-1H-indol-2-yl)carbonyl]amino}-1H-indol-2-yl)carbonyl]-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl    4-methylpiperazine-1-carboxylate hydrochloride,-   24)    (8S)-8-(chloromethyl)-6-({5-[(1H-indol-2-ylcarbonyl)amino]-1H-indol-2-yl}carbonyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl    piperazine-1-carboxylate,-   25) tert-butyl    {2-[(2-{[(3bS,4aR)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-5-yl)carbamoyl]-1H-indol-5-yl}carbamate,-   26)    5-amino-N-(2-{[(3bS,4aR)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-5-yl)-1H-indole-2-carboxamide,-   27)    (8R)-6-({5-[({5-[(tert-butoxycarbonyl)amino]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl    4-methylpiperazine-1-carboxylate,-   28)    (8R)-6-[(5-{[(5-amino-1H-indol-2-yl)carbonyl]amino}-1H-indol-2-yl)carbonyl]-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl    4-methylpiperazine-1-carboxylate hydrochloride,-   29)    N-(2-{[(3bR,4aS)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-5-yl)-5-nitro-1H-indole-2-carboxamide,-   30)    N-(2-{[(3bS,4aR)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-5-yl)-5-nitro-1H-indole-2-carboxamide,-   31)    (8S)-8-(chloromethyl)-1-methyl-6-[(5-{[(5-nitro-1H-indol-2-yl)carbonyl]amino}-1H-indol-2-yl)carbonyl]-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl    4-methylpiperazine-1-carboxylate,-   32    (8R)-8-(chloromethyl)-1-methyl-6-[(5-{[(5-nitro-1H-indol-2-yl)carbonyl]amino}-1H-indol-2-yl)carbonyl]-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl    4-methylpiperazine-1-carboxylate,-   33)    (8S)-8-(chloromethyl)-6-[(5-{[(5-hydroxy-1H-indol-2-yl)carbonyl]amino}-1H-indol-2-yl)carbonyl]-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl    4-methyl piperazine-1-carboxylate,-   34)    (8R)-8-(chloromethyl)-6-[(5-{[(5-hydroxy-1H-indol-2-yl)carbonyl]amino}-1H-indol-2-yl)carbonyl]-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl    4-methyl piperazine-1-carboxylate,-   35)    N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N˜5˜-carbamoyl-N-[4-({[{3-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]-2,2-dimethylpropyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide,-   36)    N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N˜5˜-carbamoyl-N-[4-({[{3-[({[(8R)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]-2,2-dimethylpropyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide,-   37)    [(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]{5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}methanone,-   38)    [(8R)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]{5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}methanone,-   39)    N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-[4-({[{3-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]-2,2-dimethylpropyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide,-   40)    N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-[4-({[{3-[({[(8R)-8-(chloromethyl)-1-methyl-6-({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]-2,2-dimethylpropyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide,-   41)    N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-[4-({[(3-{[({(8S)-8-(chloromethyl)-1-methyl-6-[(2E)-3-{5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}prop-2-enoyl]-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl}oxy)carbonyl](methyl)amino}-2,2-dimethylpropyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide,-   42)    N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-[4-({[(3-{[({(8R)-8-(chloromethyl)-1-methyl-6-[(2E)-3-{5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}prop-2-enoyl]-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl}oxy)carbonyl](methyl)amino}-2,2-dimethylpropyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide,-   43)    (2E)-1-[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-y]-3-(1H-indol-3-yl)prop-2-en-1-one,-   44)    N-(2-{[(8R)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-y]carbonyl}-1-methyl-1H-indol-5-yl)-1-methyl-1H-indole-2-carboxamide,-   45)    1-methyl-N-(1-methyl-2-{[(3bS,4aR)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-5-yl)-1H-indole-2-carboxamide,-   46)    N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-phenylalanyl-L-leucyl-N-[4-({[{3-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]-2,2-dimethylpropyl}(methyl)carbamoyl]oxy}methyl)phenyl]glycinamide,-   47)    N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-phenylalanyl-L-leucyl-N-[4-({[{3-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]-2,2-dimethylpropyl}(methyl)carbamoyl]oxy}methyl)phenyl]glycinamide,-   48)    N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-phenylalanyl-L-leucyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]glycinamide,-   49)    N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-phenylalanyl-L-leucyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]glycinamide,-   50)    L-valyl-N⁵-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-y]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide,-   51)    L-valyl-N⁵-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide,-   52)    N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide,-   53)    N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecan-1-oyl]-L-valyl-N⁵-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide    and-   54)    N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide.

For a reference to any specific compound of the formula (I), (II), (III)or (IV) of the invention, optionally in the form of a pharmaceuticallyacceptable salt, see the experimental section and claims.

The present invention also provides a process for the preparation of acompound of formula (I), (II), (III) or (IV) as defined above, by usingthe reaction routes and synthetic schemes described below, employing thetechniques available in the art and starting materials readilyavailable. The preparation of certain embodiments of the presentinvention is described in the examples that follow, but those ofordinary skill in the art will recognize that the preparations describedmay be readily adapted to prepare other embodiments of the presentinvention. For example, the synthesis of non-exemplified compoundsaccording to the invention may be performed by modifications apparent tothose skilled in the art, for instance by appropriately protectinginterfering groups, by changing to other suitable reagents known in theart, or by making routine modifications of reaction conditions.Alternatively other reactions referred to herein or known in the artwill be recognized as having adaptability for preparing other compoundsof the invention.

All those with ordinary skills in the art will appreciate that anytransformation performed according to said methods may require standardmodifications such as, for instance, protection of interfering groups,change to other suitable reagents known in the art, or make routinemodifications of reaction conditions.

The present invention provides a process for the preparation of acompound of formula (I) or (III) as defined above, characterized in thatthe process comprises the following step:

a) converting a compound of formula (II) or (IV)

whereinL1 is hydrogen, W1, Z1 and RM1 are null, andR1, R2, R3, R4, R6, T, BM, A′, L, W, Z and RM are as defined above,to give a compound of formula (I) or (III), respectively as definedabove, and the pharmaceutically acceptable salts thereof.

Accordingly, the preparation of a compound of formula (I) or (III) isdepicted in Scheme 1 below:

According to step a) the reaction is performed by well known proceduresreported in the art (see for example Boger, D. L.; J. Am. Chem. Soc.1996, 118, 2301-2302). An example, that is not intended to limit themethod, is the use of basic conditions such as e.g. the use of TEA,NaHCO₃ or DBU. The reaction is performed in DCM or DMF or a mixture ofthem, at a temperature ranging from 20° C. to reflux and for a timeranging from 30 minutes to about 24 hours.

The present invention also provides a process for the preparation of acompound of formula (II) as defined above, i.e. a compound of formula(II)′ wherein L1 is hydrogen and a compound of formula (II)″ wherein L1is not hydrogen, characterized in that the process comprises thefollowing steps:

b) reacting a compound of formula (XVI)

wherein R17 is halogen, OH, or an activating moiety of the carboxylicgroup, e.g. activated esters, T is as defined above and BM is a bindingmoiety of formula (V) as defined above,with a compound of formula (XVII)

wherein R18 is hydrogen or a protecting group and R1, R2, R3, R4 and R6are as defined above; optionallyc) removing the protection if present and reacting the resultantcompound of formula (II)′

wherein R1, R2, R3, R4, R6, T and BM are as defined above, with acompound of formula (XVIII)

R19-L1-W1-Z1  (XVIII)

wherein R19 is null, hydrogen, an activating moiety of the NH group,preferably tosyl, or R17, wherein R17 is as defined above, andL1, W1 and Z1 are as defined above and at least one of them is not null,to give a compound of formula (II)″

wherein L1 is as defined above except hydrogen, at least one of L1, W1and Z1 is not null,and R1, R2, R3, R4, R6, T and BM are as defined above,and the pharmaceutically acceptable salts thereof.

Accordingly, the preparation of a compound of formula (II) is depictedin Scheme 2 below:

The present invention also provides a process for the preparation of acompound of formula (IV) as defined above, i.e. a compound of formula(IV)′ wherein A′ is A and L1 is hydrogen and a compound of formula (IV)″wherein A′ is A and L1 is not hydrogen, characterized in that theprocess comprises the following steps:

d) reacting a compound of formula (XIX)

wherein A′ is A, wherein A is as defined above,T is as defined above, andBM is a binding moiety of formula (V)′ as defined above,with a compound of formula (XX)

wherein R19 is as defined above, andL, W, Z and RM are as defined above and at least one of them is notnull;e) reacting the resultant compound of formula (XXI)

wherein A′ is A, wherein A is as defined above, andT, BM, L, W, Z and RM are as defined above,with a compound of formula (XVII)

as defined above;optionallyf) reacting the resultant compound of formula (IV)′

wherein A′ is A, wherein A is as defined above,L, W, Z and RM are as defined above, and at least one of them is notnull,and R1, R2, R3, R4, R6, T and BM are as defined above,with a compound of formula (XX)′

wherein R19 is as defined above, L1 is as defined above except hydrogen,and W1, Z1 and RM1 are as defined above, and at least one of them is notnull, to give a compound of formula (IV)″

wherein L1 is as defined above except hydrogen,W1, Z1 and RM1 are as defined above, and at least one of them is notnullA′ is A, wherein A is as defined above,L, W, Z and RM are as defined above, andR1, R2, R3, R4, R6, T and BM are as defined above,and the pharmaceutically acceptable salts thereof.

The present invention also provides a process for the preparation of acompound of formula (IV) as defined above, i.e. a compound of formula(IV)′ as defined above, and a compound of formula (IVa)″ wherein A′ isA, wherein A is a saturated group selected from OH, NH₂ and COOH, and L1is not hydrogen, characterized in that the process comprises thefollowing steps:

d′) reacting a compound of formula (XIX) with a compound of formula(XVII) as defined above;e′) reacting the resultant compound of formula (XV)

wherein A′ is A, wherein A is a saturated group selected from OH, NH₂and COOHandR1, R2, R3, R4, R6, T and BM are as defined above,with the compound of formula (XX) as defined above;optionallyf) reacting the resultant compound of formula (IV)′

wherein A′ is A, wherein A is as defined above, andR1, R2, R3, R4, R6, T, BM, L, W, Z and RM are as defined abovewith a compound of formula (XX)′ defined above so to yield a compound offormula (IV)″ as defined above, and the pharmaceutically acceptablesalts.ore″) reacting the compound of formula (XV) as defined above, with thecompound of formula (XX)′ as defined above; optionallyf″) reacting the resultant compound of formula (IVa)″

wherein L1 is as defined above except hydrogen, L1, W1, Z1 and RM1 areas defined above and at least one of them is not null,A′ is A, wherein A is a saturated group selected from OH, NH₂ and COOHand R1, R2, R3, R4, R6, T and BM are as defined above,with the compound of formula (XX) as defined above, to give a compoundof formula (IV)″ as defined above, and the pharmaceutically acceptablesalts.

Accordingly, the preparation of a compound of formula (IV), i.e. acompound of formula (IV)′, (IV)″ or (IVa)″ as defined above, is depictedin Scheme 3 below:

According to step c), d), f) e′), f), e″) and f′) the coupling isperformed in a organic solvent, preferably DMF, in presence of acondensing agent such as for example DCC, EDC (for general couplingreagents see e.g. Amino Acids, Peptides and Proteins in OrganicChemistry: Building Blocks, Catalysis and Coupling Chemistry, Volume 3;Andrew B. Hughes, Ayman El-Faham, Fernando Albericio). See also specificchemical conditions reported in the experimental part below.

According to b), e), d′) the coupling reaction is preferably carried outat a temperature ranging from 20° C. to reflux, in presence optionallyof a base, and for a time ranging from 30 minutes to about 24 hours.

Compounds of formula (XVII) and (XIX) are known or can be prepared bymethods known to the expert in the art or as reported in GB2344818 citedabove or J. Med. Chem. 2003, (46) page 634-637.

Compounds of formula (XVI), (XVIII), (XX) and (XX)′ are known or can beprepared by methods known to the expert in the art or as reported inAnticancer Agents in Med Chem 2008, (8) page 618-637 or inWO2010/009124.

The present invention also provides a process for the preparation of acompound of formula (IV) as defined above, i.e. a compound of formula(IV)′″ wherein A′ is null and L1 is not hydrogen, characterized in thatthe process comprises the following step:

e′″) reacting a compound of formula (II)′

wherein A′ is null,R₁, R₂, R₃, R₄, R₆ and T are as defined above andBM is a binding moiety of formula (V) as defined above,with a compound of formula (XX)′

wherein L1 is as defined above but not hydrogen andR19, W1, Z1 and RM1 are as defined above, to give a compound of formula(IV)′″

wherein A′ is null,L1 is as defined above except hydrogen,R1, R2, R3, R4, R6, T and BM are as defined above, andW1, Z1 and RM1 are as defined above and at least one of them is notnull,and the pharmaceutically acceptable salts thereof.

Accordingly, the preparation of a compound of formula (IV), wherein A′is null, is depicted in Scheme 4 below:

According to step e′″) the coupling is performed as described under e″)above.

From all of the above, it is clear to the skilled person that whenpreparing the compounds of formula (I), (II), (III) and (IV) accordingto any one of the aforementioned process variants, optional functionalgroups within the starting materials or the intermediates thereof thatcould give rise to unwanted side reactions need to be properly protectedaccording to conventional techniques. Likewise, the conversion of theselatter into the free deprotected compounds may be carried out accordingto known procedures.

As it will be readily appreciated, if the compounds of formula (I),(II), (III) and (IV) prepared according to the process described aboveare obtained as mixture of isomers, their separation using conventionaltechniques into the single isomers of formula (I), (II), (III) and (IV)is within the scope of the present invention.

Preferably a compound of formula (II) wherein L1 is as defined aboveexcept hydrogen or

a compound of formula (IV) wherein RM and/or RM1 are not null, andR1, R2, R3, R4, R6, T, BM, A′, L, W, Z, L1, W1 and Z1 are as definedabove, is reactedg) with a compound of formula (XXII)

R17-RM  (XXII)

wherein R17 is as defined above and RM is as defined above but not null,to give the corresponding compound of formula (IV) wherein RM is asdefined above but not null,then optionallyh) with a compound of formula (XXII)′

R17-RM1  (XXII)′

wherein R17 is as defined above, and RM1 is as defined above but notnull, to give the corresponding compound of formula (IV) wherein RM1 isas defined above but not null.

It is important to underline that the steps described under g) and h)may optionally occurs in reverse order, i.e. step h) first and then stepg).

According to step g) and h) the coupling is performed as described underb) above.

Compounds of formula (XXII) and (XXII)′ are commercially available orare known compounds or can be prepared by methods known to the expert inthe art or as reported in Anticancer Agents in Med Chem 2008, (8) page618-637 or in WO2010/009124.

Preferably a compound of formula (XIX), wherein A′ is —OH and T and BMare as defined above, is reacted

i) with a compound of formula (XX)

wherein R19 is null,L is a group of formula (Xf′) or (Xg′)

wherein R9 is hydrogen, hydroxy or an optionally substituted groupselected from linear or branched C₁-C₄ alkyl, linear or branched C₁-C₄hydroxyalkyl, linear or branched C₁-C₄ sulfhydrylalkyl and linear orbranched C₁-C₄ aminoalkyl andW, Z and RM are nullorR9 is null andat least one of W, Z or RM is not null,to give a compound of formula (XI)

wherein A′ is —O—, L is a group of formula (Xf) or (Xg), T, W, Z and RMare as defined above and BM is a group of formula (V)′;orj) with a compound of formula (XX)

Wherein R19 is null,L is a group of formula (Xh′) or (Xi′)

wherein R9 is hydrogen, hydroxy or an optionally substituted groupselected from linear or branched C₁-C₄ alkyl, linear or branched C₁-C₄hydroxyalkyl, linear or branched C₁-C₄ sulfhydrylalkyl and linear orbranched C₁-C₄ aminoalkyl andW, Z and RM are nullorR9 is null andat least one of W, Z or RM is not null,to give a compound of formula (XXI)

wherein A′ is —O—, L is a group of formula (Xh) or (Xi) and T, BM, L, W,Z and RM are as defined above;ork) with a compound of formula (XX)

wherein R19 is an activating NH group preferably tosyl,L is a group of formula —NHCOR₉ (Xa), —NHCONH—R₉ (Xb), —NHCOO—R₉ (Xc),or —NH—R₉ (Xd);wherein R9 is hydrogen, hydroxy or an optionally substituted groupselected from linear or branched C₁-C₄ alkyl, linear or branched C₁-C₄hydroxyalkyl, linear or branched C₁-C₄ sulfhydrylalkyl and linear orbranched C₁-C₄ aminoalkyl andW, Z and RM are nullorR9 is null andat least one of W, Z or RM is not null,to give a compound of formula (XXI)

wherein A′ is —O—, L is a group of formula (Xa) to (Xd) and T, BM, W, Zand RM are as defined above;orl) with a compound of formula of formula (XX)

wherein R19 is R17 wherein R17 is —OH,L is a group of formula (Xe)

whereinR9 and R10 are, each independently, hydrogen, hydroxy or an optionallysubstituted group selected from linear or branched C₁-C₄ alkyl, linearor branched C₁-C₄ hydroxyalkyl, linear or branched C₁-C₄ sulfhydrylalkyland linear or branched C₁-C₄ aminoalkyl andW, Z and RM are nullorone of R9 or R10 is null andat least one of W, Z or RM is not null,to give a compound of formula (XXI)

wherein A′ is —O—, L is a group of formula (Xe) and T, BM, W, Z and RMare as defined above.

According to step i) and j) the reaction is carried in an organicsolvent, preferably DCM or DMF, in presence of PTSA at a temperatureranging from 20° C. to reflux and for a time ranging from 30 minutes toabout 24 hours. Removal of the protecting group is performed using knownprocedure reported in the literature (see e.g. Protective Groups inOrganic Synthesis; Theodora W. Greeen, Peter G. M. Wuts).

According to step k) the reaction is performed in a organic solvent,preferably ether, dioxane or a mixture of them with LiHMDS at atemperature ranging from −10° C. to 50° C. and for a time ranging from30 minutes to about 24 hours.

Removal of the protecting group is performed using known procedurereported in the literature (see e.g. Protective Groups in OrganicSynthesis; Theodora W. Greeen, Peter G. M. Wuts).

According to step l) the reaction is performed in an organic solvent,preferably DCM, THF, CH₃CN or CCl₄, optionally in presence of a base,preferably DIPEA, at a temperature ranging from −10° C. to 50° C. andfor a time ranging from 30 minutes to about 24 hours.

Preferably a compound of formula (XIX) wherein A′ is —OH or —NH₂, and Tand BM are as defined above, is reacted

m) with a compound of formula (XX)

wherein R19 is R17 wherein R17 is an activating moiety of the carboxylicgroup, preferably pyrrolidin-2,5-dione-1yl, L is a group of formula (Xj)or (Xk)

wherein R9 is hydrogen, hydroxy or an optionally substituted groupselected from linear or branched C₁-C₄ alkyl, linear or branched C₁-C₄hydroxyalkyl, linear or branched C₁-C₄ sulfhydrylalkyl and linear orbranched C₁-C₄ aminoalkyl andW, Z and RM are nullorR9 is null andat least one of W, Z or RM is not null,to give a compound of formula (XXI)

wherein A′ is —O— or —NH, L is a group of formula (Xj) or (Xk) and T,BM, W, Z and RM are as defined above.

According to step m) the coupling reaction is performed in a organicsolvent, preferably DCM, in basic conditions, e.g. TEA, DMAP. Thereaction is carried out at a temperature ranging from 0° C. to refluxand for a time ranging from 30 minutes to about 24 hours.

Preferably a compound of formula (XIX) wherein A′ is —COOH, and T and BMare as defined above, is reacted

n) with a compound of formula (XX)

wherein R19 is hydrogen,L is a group of formula (Xm) or (Xn)

whereinR9 and R10 are, each independently, hydrogen, hydroxy or an optionallysubstituted group selected from linear or branched C₁-C₄ alkyl, linearor branched C₁-C₄ hydroxyalkyl, linear or branched C₁-C₄ sulfhydrylalkyland linear or branched C₁-C₄ aminoalkyl andW, Z and RM are nullorone of R9 or R10 is null andat least one of W, Z or RM is not null,to give a compound of formula (XXI)

wherein A′ is —CO—, L is a group of formula (Xm) or (Xn) and T, BM, W, Zand RM are as defined above;oro) with a compound of formula (XX)

wherein L is null,W is a group of formula (XIa) to (XIe), (XIh) to (XIj)

wherein R9 is hydrogen,R10 is hydrogen, hydroxy or an optionally substituted group selectedfrom linear or branched C₁-C₄ alkyl, linear or branched C₁-C₄hydroxyalkyl, linear or branched C₁-C₄ sulfhydrylalkyl and linear orbranched C₁-C₄ aminoalkyl andW, Z and RM are nullorR10 is null andat least one of W, Z or RM is not null,R11 and R12 are as defined above,A₁ is —CH₂—, —CH₂N(R12)- or —NR12-, wherein R12 is as defined above,to give a compound of formula (XXI)

wherein A′ is —CO—, L is null, W is a group of formula (XIa) to (XIe),(XIh) and (XIj), T, BM, W, Z and RM are as defined above.

According to step n) the reaction is performed in a organic solvent,preferably DCM, in basic conditions e.g. TEA and optionally in presenceof a condensing agent such as for example DCC, EDC, at a temperatureranging from 0° C. to reflux and for a time ranging from 30 minutes toabout 24 hours.

According to step o) the coupling reaction is performed using conditionswell known in the literature (see e.g. Scott, C. J. et al. J. Med. Chem.2005, 48, 1344-1358; Amino Acids, Peptides and Proteins in OrganicChemistry: Building Blocks, Catalysis and Coupling Chemistry, Volume 3;Andrew B. Hughes, Ayman El-Faham, Fernando Albericio). Preferably acompound of formula (XIX) wherein A′ is —OH or —NH₂, and T and BM asdefined above, is reacted

p) with a compound of formula (XX)

wherein R19 is null,L is null,W is a group of formula (XIa) to (XIj)

wherein R10 is —OH,R9 is hydrogen, hydroxy or an optionally substituted group selected fromlinear or branched C₁-C₄ alkyl, linear or branched C₁-C₄ hydroxyalkyl,linear or branched C₁-C₄ sulfhydrylalkyl and linear or branched C₁-C₄aminoalkyl andW, Z and RM are nullorR9 is null andat least one of W, Z or RM is not null,R11, R12, m and A₁ are as defined above,to give a compound of formula (XXI)

wherein A′ is —O— or NH—, L is null, W is a group of formula (XIa) to(XIi), T, BM, W, Z and RM are as defined above.

According to step p) the coupling reaction is performed as describedunder o) above.

Steps i) to m) and p) can also be used to convert compounds of formula(IV)′ to compounds of formula (IV)″ by reaction with compounds offormula (XX)′ (step f) or to convert compounds of formula (XV) tocompounds of formula (IV)′″ by reaction with compounds of formula (XX)′(step e″) or to convert compounds of formula (II)′ to compounds offormula (II)″ by reaction with compounds of formula (XVIII) (step c) orto convert compounds of formula (XV) to compounds of formula (IV)′ byreaction with compounds of formula (XX) (step e′) or to convertcompounds of formula (IV)′″ to compounds of formula (IV)″ by reactionwith compounds of formula (XX) (step f″).

Pharmacology

The compounds of the present invention are useful as antitumor agents.

A mammal, e.g. a human or animal, may therefore be treated by a methodcomprising administering thereto a pharmaceutically effective amount ofa compound of formula (I), (II), (III) or (IV).

The condition of the human or animal may be ameliorated or improved inthis way.

The evaluation of the cytotoxicity of the compounds of formula (I),(II), (III), or (IV) is assessed as described below.

In Vitro Cell Proliferation Assay

A2780 human ovarian and MCF7 human breast cancer cells (1250 cells/well)were seeded in white 384 well-plates in complete medium (RPMI1640 orEMEM plus 10% Fetal bovine serum) and treated with compounds dissolvedin 0.1% DMSO, 24 h after seeding. The cells were incubated at 37° C. and5% CO₂ and after 72 h the plates were processed using CellTiter-Gloassay (Promega) following the manufacturer's instruction.

CellTiter-Glo is a homogenous method based on the quantification of theATP present, an indicator of metabolically active cells. ATP isquantified using a system based on luciferase and D-luciferin resultinginto light generation. The luminescent signal is proportional to thenumber of cells present in culture.

Briefly, 25 μL/well of reagent solution are added to each well and after5 minutes shaking microplates are read by a luminometer. The luminescentsignal is proportional to the number of cells present in culture.

Representative compounds of the invention of formula (I) or (II) weretested in the specific in vitro cell proliferation assay describedabove.

All the tested compounds have an IC₅₀ value <0.5 μM (<500 nM) in A2780human ovarian cancer cells.

In particular, Compd. 7, 9, 23, 6, 1 and 5 have IC₅₀ value <50 nM;Compd. 22, 17 and 20 have an IC₅₀ value <0.1 nM; Compd. 18, 8 and 37have an 1050 value <0.01 nM.

As can be appreciated by the skilled person, all these representativecompounds are thus particularly advantageous in antitumor therapy.

Furthermore the functionalized compounds of formula (III) or (IV) of thepresent invention are suitable to be conjugated.

The ability of the functionalized derivatives of formula (III) or (IV)to be conjugated has been assessed by conjugating them with anucleophilic group such as the SH group of the cysteine aminoacid.

Preparation of a Conjugate

2 nmol of cysteine (MW 121 Da) have been reacted with 2 nmol of afunctionalized compound of formula (IV), i.e. Compd. 54 (MW 1221Da).

The reaction was incubated for 1 h at 21° C. in presence of Boratebuffer 50 mM pH8, DTPA 2 mM, NaCl 50 mM, obtaining conjugate A1(m/z=1343 (MH+)) then was analyzed by HPLC ESI-MS using a reversed phaseHPLC method (PLRP-S column 1000A 8 uM 150×2.1 mm) on a 1100 Agilent HPLCinstrument coupled with an Agilent 1946 single quadrupole massspectrometry detector with an orthogonal ESI source.

FIG. 1 shows the mass spectrum of the conjugate A1 and reports themolecular weight (m/z) on the x axis while intensity expressed as countsper second (cps) is reported on the y axis.

FIG. 2 shows the HPLC profile of the conjugate A1 and reports the time(min) on the x axis while UV absorbance (mAU) is reported on the y axis.

Release of a Drug Moiety from a Conjugate

As an example, that is not intended to limit the scope of the invention,the release of a compound of formula (II) from the conjugate wasperformed in presence of cathepsin as reported below.

The conjugate A1 was incubated with 0.2 unit of cathepsin B in sodiumacetate buffer pH 5.5 and 1 mM Cys for 2 hours at 40° C.

Disappearance of the conjugate A1 and release of the correspondingcompound of formula (II), i.e. compd. 37, as well as of its precursorA3, confirms the breaking of the Z peptidic linker of the conjugate.

Complete release of the compound of formula (II) from the conjugate hasbeen observed by HPLC ESI-MS analysis.

FIG. 3 shows the HPLC profile after 2 h treatment of conjugate A1 withcathepsin and reports the time (min) on the x axis while UV absorbance(mAU) is reported on the y axis.

FIG. 4 a shows the mass spectrum of the released Compd. 37 and reportsthe molecular weight (m/z) on the x axis while intensity expressed ascounts per second (cps) is reported on the y axis.

FIG. 4 b shows the mass spectrum of the released Compound A3, aprecursor of 37, and reports the molecular weight (m/z) on the x axiswhile intensity expressed as counts per second (cps) is reported on they axis.

The compounds of the present invention can be administered either assingle agents or, alternatively, in combination with known anticancertreatments such as radiation therapy or chemotherapy regimen, incombination with cytostatic or cytotoxic agents, antibiotic-type agents,alkylating agents, antimetabolite agents, hormonal agents, immunologicalagents, interferon-type agents, cyclooxygenase inhibitors (e.g. COX-2inhibitors), matrix metalloprotease inhibitors, telomerase inhibitors,tyrosine kinase inhibitors, anti-growth factor receptor agents, anti-HERagents, anti-EGFR agents, anti-angiogenesis agents (e.g. angiogenesisinhibitors), farnesyl transferase inhibitors, ras-raf signaltransduction pathway inhibitors, cell cycle inhibitors, other cdksinhibitors, tubulin binding agents, topoisomerase I inhibitors,topoisomerase II inhibitors, and the like.

If formulated as a fixed dose, such combination products employ thecompounds of this invention within the dosage range described below andthe other pharmaceutically active agent within the approved dosagerange.

Compounds of formula (I), (II), (III) or (IV) may be used sequentiallywith known anticancer agents when a combination formulation isinappropriate.

The compounds of formula (I), (II), (III) or (IV) of the presentinvention, suitable for administration to a mammal, e.g., to humans, canbe administered by the usual routes and the dosage level depends uponthe age, the weight, the conditions of the patient and theadministration route.

For example, a suitable dosage adopted for oral administration of acompound of formula (I) may range from about 1 to about 300 mg per dose,from 1 to 5 times daily. The compounds of the invention can beadministered in a variety of dosage forms, e.g., orally, in the form oftablets, capsules, sugar or film coated tablets, liquid solutions orsuspensions; rectally in the form of suppositories; parenterally, e.g.,subcutaneous, intramuscularly, or through intravenous and/or intrathecaland/or intraspinal injection or infusion.

The present invention also includes pharmaceutical compositionscomprising a compound of formula (I) or a pharmaceutically acceptablesalt thereof in association with a pharmaceutically acceptableexcipient, which may be a carrier or a diluent.

The pharmaceutical compositions containing the compounds of theinvention are usually prepared following conventional methods and areadministered in a suitable pharmaceutical form. For example, the solidoral forms may contain, together with the active compound, diluents,e.g., lactose, dextrose, saccharose, cellulose, corn starch or potatostarch; lubricants, e.g., silica, talc, stearic acid, magnesium orcalcium stearate, and/or polyethylene glycols; binding agents, e.g.,starches, arabic gum, gelatine methylcellulose, carboxymethylcelluloseor polyvinyl pyrrolidone; disintegrating agents, e.g., starch, alginicacid, alginates or sodium starch glycolate; effervescing mixtures;dyestuffs; sweeteners; wetting agents such as lecithin, polysorbates,laurylsulphates; and, in general, non-toxic and pharmacologicallyinactive substances used in pharmaceutical formulations. Thesepharmaceutical preparations may be manufactured in known manner, forexample, by means of mixing, granulating, tabletting, sugar-coating, orfilm-coating processes.

The liquid dispersions for oral administration may be, e.g., syrups,emulsions and suspensions. As an example, the syrups may contain, ascarrier, saccharose or saccharose with glycerine and/or mannitol andsorbitol.

The suspensions and the emulsions may contain, as examples of carriers,natural gum, agar, sodium alginate, pectin, methylcellulose,carboxymethylcellulose or polyvinyl alcohol. The suspension or solutionsfor intramuscular injections may contain, together with the activecompound, a pharmaceutically acceptable carrier, e.g. sterile water,olive oil, ethyl oleate, glycols, e.g. propylene glycol, and, ifdesired, a suitable amount of lidocaine hydrochloride. The solutions forintravenous injections or infusions may contain, as a carrier, sterilewater or preferably they may be in the form of sterile, aqueous,isotonic, saline solutions or they may contain propylene glycol as acarrier. The suppositories may contain, together with the activecompound, a pharmaceutically acceptable carrier, e.g. cocoa butter,polyethylene glycol, a polyoxyethylene sorbitan fatty acid estersurfactant or lecithin.

With the aim at better illustrating the present invention, withoutposing any limitation to it, the following examples are now given.

EXAMPLES

The synthetic preparation of some compounds of formula (I) of theinvention is described in the following examples. The compounds of thepresent invention, as prepared according to the following examples, werealso characterized by ¹H-NMR and/or by Exact mass data ESI(+).

¹H-NMR spectra were recorded at a constant temperature of 25° C. on aVarian INOVA 500 spectrometer (operating at 499.8 MHz for ¹H, 125.8 for¹³C and 50.6 MHz for ¹⁵N) and equipped with 5 mm ¹H{¹³c, ¹⁵N} z axis PFGIndirect Detection Cold-Probe or alternatively with 5 mm ¹H{¹³C—¹⁵N} zaxis PFG Triple Resonanace Probe.

Chemical shifts were referenced with respect to the residual solventssignals. Data are reported as follows: chemical shift (δ), multiplicity(s=singlet, d=doublet, t=triplet, q=quartet, br. s=broad singlet,td=triplet of doublet, dd=doublet of doublets, ddd=doublet of doubletsof doublets, m=multiplet), coupling constants (Hz), and number ofprotons.

Exact mass data ESI(+) were obtained on a Waters Q-T of Ultima massspectrometer directly connected with a Agilent 1100 micro-HPLC system aspreviously described (M. Colombo, F. Riccardi-Sirtori, V. Rizzo, RapidCommun. Mass Spectrom. 2004, 18, 511-517).

In the examples below as well as throughout the application, thefollowing abbreviations have the following meanings. If not defined, theterms have their generally accepted meanings.

ABBREVIATIONS DCC N,N′-dicyclohexylcarbodiimide DBU diazabicycloundeceneDCM dichloromethane DIPEA N,N-diisopropyethylamine DMAPN,N-dimethylaminopyridine DMF N,N-dimethylformamide DMSO dimethylsulfoxide EDCI N-ethyl-N′,N′-diisopropyl carbodiimide hydrochloride EDC1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Et₂O diethyl ether EtOAcethyl acetate EtOH ethanol HCl hydrochloric acid HOBt1H-benzotriazol-1-ol LiHMDS lithium bis(trimethylsilyl)amide MeOHmethanol Na₂SO₄ sodium sulfate NaHCO₃ sodium hydrogen carbonate NaOHsodium hydroxide TEA triethylamine TFA trifluoro acetic acid THFtetrahydrofurane

Example 1 Step b, Step aN-(6-{[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1H-indol-3-yl)-1H-indole-6-carboxamide[(II)] (Compd. 1)

Step b

A solution of(8S)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-ol(XVII) prepared as reported in the GB2344818 (14.2 mg, 0.0563 mmol) wasdissolved in dry DMF (1.5 mL), and treated with EDCI (43 mg, 4 eq.) and3-[(1H-indol-6-ylcarbonyl)amino]-1H-indole-6-carboxylic acid (XVI) (27mg, 1.5 eq.) The mixture was stirred for 16 h at room temperature andthen was quenched by adding saturated aqueous NaCl. Isolation of theproduct was performed by extraction with EtOAc (×4) and subsequentwashing of the combined organic layers with aqueous 2M HCl (×3),saturated aqueous Na₂CO₃ (×3) and saturated aqueous NaCl (×3). Organiclayer was dried (Na₂SO₄), concentrated under vacuum. The crude residuewas purified by flash chromatography (hexane-acetone 6:4) to afford thetitle compound (18.7 mg, 60%).

ESI MS: m/z 555 (MH+)

Analogously, by using the corresponding carboxylic acids, the followingcompounds have been prepared:

N-(5-{[(8R)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1-methyl-1H-pyrrol-3-yl)-1-methyl-4-{[(1-methyl-4-nitro-1H-pyrrol-2-yl)carbonyl]amino}-1H-pyrrole-2-carboxamide[(II)] (Compd. 2)

ESI MS: m/z 650 (MH+)

(2E)-1-[(8R)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)prop-2-en-1-one[(II)] (Compd. 3)

ESI MS: m/z 424 (MH+)

(2E)-1-[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)prop-2-en-1-one[(II)] (Compd. 4)

ESI MS: m/z 424 (MH+)

N-(3-{(1E)-3-[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]-3-oxoprop-1-en-1-yl}-1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-indole-2-carboxamide[(II)] (Compd. 13)

ESI MS: m/z 582 (MH+)

N-(3-{(1E)-3-[(8R)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]-3-oxoprop-1-en-1-yl}-1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-indole-2-carboxamide[(II)] (Compd. 14)

ESI MS: m/z 582 (MH+)

(2E)-1-[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]-3-(1H-indol-3-yl)prop-2-en-1-one[(II)] (Compd. 43)

ESI MS: m/z 423 (MH+)

¹H NMR (500 MHz, acetone-d6) δ ppm 2.60 (d, J=1.0 Hz, 3H) 3.58 (m, 1H)3.88 (m, J=11.2, 1.9 Hz, 1H) 4.2 (m, 1H) 4.46 (t, J=9.5 Hz, 1H) 4.63 (d,J=10.6 Hz, 1H) 7.07 (d, J=15.4 Hz, 1H) 7.24 (m, 2H) 7.32 (m, 1H) 7.53(m, 1H) 7.91 (m, 1H) 8.02 (m, 2H) 8.13 (br. s., 1H) 9.26 (br. s., 1H)10.81 (br. s., 1H)

N-(2-{[(8R)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1-methyl-1H-indol-5-yl)-1-methyl-1H-indole-2-carboxamide[(II)] (Compd. 44)

ESI MS: m/z 583 (MH+)

Step aN-(6-{[(3bR,4aS)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-3-yl)-1H-indole-6-carboxamide[(I)] (Compd. 5)

Compound 1 (25 mg, 0.045 mmol) was dissolved in DMF (2 mL), and treatedwith a solution of NaHCO₃ in water (1 mL, 15 mg NaHCO₃/mL). The reactionmixture was stirred for 2 h, EtOAc was added and the resulting organiclayer was washed with brine (×4), dried (Na₂SO₄) and concentrated invacuo. The crude residue was purified by flash chromatography(hexane-acetone 6:4) to afford compound 5 (19 mg, 82%).

ESI MS: m/z 519 (MH+)

¹H NMR (500 MHz, acetone-d₆) δ ppm 1.75 (t, J=4.8 Hz, 1H) 2.27 (s, 3H)2.32 (dd, J=7.6, 4.6 Hz, 1H) 3.40 (dt, J=7.5, 4.9 Hz, 1H) 4.13 (d,J=11.3 Hz, 1H) 4.30 (dd, J=11.1, 4.7 Hz, 1H) 5.78 (s, 1H) 6.57 (br. s.,1H) 7.32 (dd, J=8.4, 0.7 Hz, 1H) 7.44 (s, 1H) 7.52 (t, J=2.7 Hz, 1H)7.67 (d, J=8.2 Hz, 1H) 7.79 (m, 2H) 8.00 (d, J=8.2 Hz, 1H) 8.22 (d,J=2.7 Hz, 2H) 9.51 (s, 1H) 10.45 (br. s, 1H) 10.61 (br. s, 1H)

Analogously, by using the corresponding carboxylic acids, the followingcompounds have been prepared:

1-methyl-N-(1-methyl-5-{[(3bS,4aR)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-pyrrol-3-yl)-4-{[(1-methyl-4-nitro-1H-pyrrol-2-yl)carbonyl]amino}-1H-pyrrole-2-carboxamide[(I)] (Compd. 6)

ESI MS: m/z 614 (MH+)

¹H NMR (500 MHz, DMSO-d₆) δ ppm 1.52 (t, J=4.9 Hz, 1H) 2.21 (s, 3H) 2.28(dd, J=7.6, 4.3 Hz, 1H) 3.43 (dt, J=7.8, 5.2 Hz, 1H) 3.76 (s, 3H) 3.86(s, 3H) 3.96 (s, 3H) 4.16 (d, J=10.7 Hz, 1H) 4.29 (dd, J=10.6, 4.8 Hz,1H) 6.30 (s, 1H) 6.77 (d, J=1.7 Hz, 1H) 7.08 (d, J=1.8 Hz, 1H) 7.25 (d,J=1.7 Hz, 1H) 7.48 (d, J=1.7 Hz, 1H) 7.58 (t, J=1.9 Hz, 2H) 8.19 (d,J=1.8 Hz, 1H) 10.00 (s, 1H) 10.30 (s, 1H)

(3bS,4aR)-3-methyl-6-[(2E)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)prop-2-enoyl]-4,4-a,5,6-tetrahydro-8H-cyclopropa[c]thieno[3,2-e]indol-8-one[(I)] (Compd. 7)

ESI MS: m/z 388 (MH+)

¹H NMR (500 MHz, acetone-d₆) δ ppm 1.45 (t, J=4.8 Hz, 1H) 2.19 (dd,J=7.6, 4.5 Hz, 1H) 2.26 (d, J=0.7 Hz, 3H) 3.44 (dt, J=7.7, 4.8 Hz, 1H)4.40 (dd, J=10.4, 4.8 Hz, 1H) 4.51 (d, J=10.3 Hz, 1H) 6.98 (d, J=15.4Hz, 1H) 7.08 (br. s., 1H) 7.24 (dd, J=7.9, 4.7 Hz, 1H) 7.44 (s, 1H) 7.98(d, J=15.4 Hz, 1H) 8.09 (s, 1H) 8.34 (dd, J=4.8, 1.5 Hz, 1H) 8.41 (dd,J=7.8, 1.3 Hz, 1H) 11.30 (s, 1H)

(3bR,4aS)-3-methyl-6-[(2E)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)prop-2-enoyl]-4,4-a,5,6-tetrahydro-8H-cyclopropa[c]thieno[3,2-e]indol-8-one[(I)] (Compd. 8)

ESI MS: m/z 388 (MH+)

¹H NMR (500 MHz, acetone-d₆) δ ppm 1.45 (t, J=4.9 Hz, 1H) 2.16-2.21 (m,1H) 2.26 (s, 3H) 3.44 (dt, J=7.7, 4.9 Hz, 1H) 4.40 (dd, J=10.4, 5.1 Hz,1H) 4.51 (d, J=10.4 Hz, 1H) 6.98 (d, J=15.4 Hz, 1H) 7.08 (br. s., 1H)7.24 (dd, J=7.8, 4.5 Hz, 1H) 7.44 (s, 1H) 7.98 (d, J=15.4 Hz, 1H) 8.09(s, 1H) 8.34 (dd, J=4.7, 1.1 Hz, 1H) 8.41 (d, J=7.8 Hz, 1H) 11.29 (br.s., 1H)

N-(3-{(1E)-3-[(3bR,4aS)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]-3-oxoprop-1-en-1-yl}-1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-indole-2-carboxamide[(I)] (Compd. 15)

ESI MS: m/z 546 (MH+)

N-(3-{(1E)-3-[(3bR,4aS)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]-3-oxoprop-1-en-1-yl}-1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-indole-2-carboxamide[(I)] (Compd. 16)

ESI MS: m/z 546 (MH+)

¹H NMR (500 MHz, acetone-d₆) δ ppm 1.58 (m, 1H) 2.24 (m, 1H) 2.27 (s,3H) 3.47 (dt, J=7.6, 4.9 Hz, 1H) 4.39 (dd, J=10.2, 4.9 Hz, 1H) 4.48 (m,1H) 6.96 (d, J=15.1 Hz, 1H) 7.01 (s, 1H) 7.12 (m, 2H) 7.28 (m, 1H) 7.40(d, J=1.8 Hz, 1H) 7.45 (s, 1H) 7.61 (d, J=8.3 Hz, 1H) 7.70 (d, J=8.1 Hz,1H) 7.98 (m, 1H) 8.09 (d, J=2.8 Hz, 1H) 8.76 (d, J=2.0 Hz, 1H) 8.87 (d,J=2.0 Hz, 1H) 9.76 (br. s., 1H) 10.97 (br. s., 1H) 11.32 (br. s., 1H)

1-methyl-N-(1-methyl-2-{[(3bS,4aR)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-5-yl)-1H-indole-2-carboxamide[(I)] (Compd. 45)

ESI MS: m/z 547 (MH+)

¹H NMR (500 MHz, acetone-d6) δ ppm 1.65 (t, J=4.9 Hz, 1H) 2.27 (d,J=12.4 Hz, 1H) 2.27 (d, J=0.7 Hz, 3H) 3.43 (dt, J=7.6, 4.89 Hz, 1H) 3.93(s, 3H) 4.12 (s, 3H) 4.38 (d, J=11.1 Hz, 1H) 4.51 (dd, J=10.8, 4.8 Hz,1H) 6.52 (s, 1H) 7.08 (s, 1H) 7.13 (m, 1H) 7.26 (s, 1H) 7.32 (td, J=7.7,1.0 Hz, 1H) 7.47 (d, J=1.0 Hz, 1H) 7.54 (m, 2H) 7.66 (d, J=7.8 Hz, 1H)7.71 (dd, J=9.1, 2.0 Hz, 1H) 8.29 (d, J=2.0 Hz, 1H) 9.54 (s, 1H)

Example 2 tert-butyl{2-[(2-{[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1H-indol-5-yl)carbamoyl]-1H-indol-5-yl}carbamate[(II)] (Compd. 18) Step b, Deprotection, Step a

Step b

A solution of(8S)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-ol((XVII), 11.4 mg, 0.045 mmol), prepared as reported in GB2344818, wasdissolved in dry DMF (1 mL), and treated with EDCI (35 mg, 4 eq.) and5-[({5-[(tert-butoxycarbonyl)amino]-1H-indol-2-yl}carbonyl)amino]-1H-indole-2-carboxylicacid (XVI) (29 mg, 1.5 eq.) The mixture was stirred for 16 h at roomtemperature and then was quenched by adding saturated aqueous NaCl.Isolation of the product was performed by extraction with EtOAc (×4) andsubsequent washing of the combined organic layers with aqueous 2M HCl(×3), saturated aqueous Na₂CO₃ (×3) and saturated aqueous NaCl (×3).Organic layer was dried (Na₂SO₄), concentrated under vacuum to give thetitle compd. 18, that is then purified by flash chromatography(hexane-acetone 1:1).

ESI MS: m/z 670 (MH+)

¹H NMR (500 MHz, acetone-d₆) δ ppm 1.50 (s, 9H) 1.79 (dt, J=6.4, 3.3 Hz,1H) 2.61 (s, 2H) 3.53-3.66 (m, 1H) 3.89 (dd, J=11.4, 2.8 Hz, 1H) 4.25(m, 1H) 4.73 (m, 1H) 4.84 (d, J=10.6 Hz, 1H) 7.21 (s, 1H) 7.26 (s, 1H)7.34 (m, 2H) 7.49 (d, J=8.8 Hz, 1H) 7.56 (m, 1H) 7.61 (m, 1H) 7.96 (br.s., 2H) 8.21 (br. s., 1H) 8.34 (s, 1H) 9.28 (s, 1H) 9.52 (s, 1H) 10.73(br. s., 1H) 10.80 (br. s., 1H).

By deprotection, the following compound has been obtained:

5-amino-N-(2-{[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-5a,7,8,8a-tetrahydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1H-indol-5-yl)-1H-indole-2-carboxamide[(II)] (Compd. 19)

A solution of compd. 18 (18 mg, 0.0268 mmol) in 3.5 M HCl-EtOAc (5 mL)was stirred for 30 minutes before removing the solvent under a steadystream of nitrogen and affording hydrochloride derivative of compd. 2(15 mg, 89%).

ESI MS: m/z 570 (MH⁺)

Step a tert-butyl{2-[(2-{[(3bR,4aS)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-5-yl)carbamoyl]-1H-indol-5-yl}carbamate[(I)] (Compd. 20)

Compd. 18 (30 mg, 0.045 mmol) was dissolved in DMF (1 mL), and treatedwith aqueous NaHCO₃ (0.5 mL, 15 mg NaHCO₃/mL). The reaction mixture wasstirred for 2 h, EtOAc was added and the resulting organic layer waswashed with brine (×4), dried (Na₂SO₄) and concentrated in vacuo. Thecrude residue was purified by flash chromatography (hexane-acetone 6:4)to afford the final compd. 20 (9 mg, 31%).

ESI MS: m/z 634 (MH⁺)

¹H NMR (500 MHz, acetone-d₆) δ ppm 1.41 (s, 9H) 1.56 (t, J=4.8 Hz, 1H)2.22 (m, 1H) 2.29 (d, J=0.9 Hz, 3H) 3.52 (m, 1H) 4.69 (m, 2H) 7.06 (s,1H) 7.26 (d, J=1.5 Hz, 1H) 7.28 (d, J=1.8 Hz, 1H) 7.36 (dd, J=8.8, 1.8Hz, 1H) 7.48 (m, 1H) 7.49 (d, J=8.8 Hz, 1H) 7.58 (m, 1H) 7.63 (m, 1H)7.96 (br. s., 1H) 8.26 (br. s., 1H) 8.35 (s, 1H) 9.55 (s, 1H) 10.77 (br.s., 1H) 10.95 (br. s., 1H)

By deprotection the following compound has been prepared:

5-amino-N-(2-{[(3bR,4aS)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-5-yl)-1H-indole-2-carboxamide[(I)] (Compd. 21)

ESI MS: m/z 534 (MH⁺)

By analogue procedure and using the suitable starting material thefollowing compounds have been prepared:

tert-butyl{2-[(2-{[(3bS,4aR)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-5-yl)carbamoyl]-1H-indol-5-yl}carbamate[(I)] (Compd. 25)

ESI MS: m/z 634 (MH⁺)

5-amino-N-(2-{[(3bS,4aR)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-5-yl)-1H-indole-2-carboxamide[(I)] (Compd. 26)

ESI MS: m/z 534 (MH⁺)

N-(2-{[(3bR,4aS)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-5-yl)-5-nitro-1H-indole-2-carboxamide[(I)] (Compd. 29)

ESI MS: m/z 564 (MH⁺)

N-(2-{[(3bS,4aR)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-5-yl)-5-nitro-1H-indole-2-carboxamide[(I)] (Compd. 30)

ESI MS: m/z 564 (MH⁺)

Example 3 Step b, Deprotection, Step a

Step b The Intermediate(8R)—N-(5-{[5-({5-[(3-amino-3-oxopropyl)carbamoyl]-1-methyl-1H-pyrrol-3-yl}carbamoyl)-1-methyl-1H-pyrrol-3-yl]carbamoyl}-1-methyl-1H-pyrrol-3-yl)-4-(benzyloxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6-carboxamide

A solution of4-amino-N-[5-({5-[(3-amino-3-oxopropyl)carbamoyl]-1-methyl-1H-pyrrol-3-yl]carbamoyl)-1-methyl-1H-pyrrol-3-yl]-1-methyl-1H-pyrrole-2-carboxamide,prepared as reported in J. Med. Chem. 2004, 47, 2611-2623, (155 mg,0.341 mmol), triethylamine (0.048 mL, 0.341 mmol) and CDI (300 mg, 1.71mmol) in dry DMF (5 mL) was stirred at room temperature for 1 h. Afterevaporation of the solvent, the residue was treated with THF and stirredovernight. The precipitate (comp XVI) was filtered and dried undervacuum at 50° C. and treated with(8R)-4-(benzyloxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole(comp (XVII) (15.5 mg, 0.045 mmol, prepared as reported in theGB2344818) in dry DMF (2 mL) in the presence of NaHCO₃ (4 mg, 0.047mmol) and were stirred under nitrogen atmosphere at room temperature for16 h. After evaporation of the solvent the residue was purified by flashchromatography (DCM-MeOH 95:5) to afford the intermediate (30 mg, 80%).

ESI MS: m/z 824 (MH+)

¹H NMR (500 MHz, DMSO-d₆) δ ppm 2.31 (t, J=7.2 Hz, 2H) 2.53 (s, 3H) 3.53(t, J=10.4 Hz, 1H) 3.80 (s, 3H) 3.85 (s, 3H) 3.87 (s, 3H) 4.12 (m, 1H)4.18 (m, 1H) 4.32 (d, J=10.4 Hz, 1H) 5.27 (s, 2H) 6.83 (d, J=1.7 Hz, 1H)6.84 (m, 1H) 7.05 (m, 2H) 7.13 (d, J=1.5 Hz, 1H) 7.20 (d, J=1.5 Hz, 1H)7.25 (d, J=1.5 Hz, 1H) 7.36 (m, 2H) 7.41 (s, 1H) 7.43 (m, 2H) 7.50 (d,J=7.3 Hz, 2H) 7.99 (m, 2H) 8.77 (s, 1H) 9.91 (s, 1H) 9.93 (s, 1H)

Analogously, by using the corresponding acyl derivative, the followingcompounds have been prepared:

(8S)—N-(5-{[5-({5-[(3-amino-3-oxopropyl)carbamoyl]-1-methyl-1H-pyrrol-3-yl}carbamoyl)-1-methyl-1H-pyrrol-3-yl]carbamoyl}-1-methyl-1H-pyrrol-3-yl)-4-(benzyloxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6-carboxamide

ESI MS: m/z 824 (MH+)

N-(2-{[(8S)-4-(benzyloxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1H-indol-5-yl)-5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indole-2-carboxamide

ESI MS: m/z 758 (MH+)

¹H NMR (500 MHz, acetone-d₆) δ ppm 1.74 (m, 4H) 2.59 (m, 4H) 2.63 (d,J=1.0 Hz, 3H) 2.86 (m, 2H) 3.65 (dd, J=11.4, 9.6 Hz, 1H) 3.93 (dd,J=11.4, 2.4 Hz, 1H) 4.12 (t, J=6.1 Hz, 2H) 4.29 (m, 1H) 4.77 (m, 1H)4.87 (m, 1H) 5.36 (m, 2H) 6.94 (dd, J=8.8, 2.3 Hz, 1H) 7.15 (d, J=2.3Hz, 1H) 7.25 (m, 2H) 7.35 (m, 1H) 7.38 (d, J=1.0 Hz, 1H) 7.43 (m, 2H)7.49 (d, J=9.1 Hz, 1H) 7.58 (m, 3H) 7.63 (m, 1H) 8.18 (br. s., 1H) 8.37(d, J=1.3 Hz, 1H) 9.52 (s, 1H) 10.77 (br. s., 1H) 10.87 (br. s., 1H)

[(8S)-4-(benzyloxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]{5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}methanonehydrochloride

ESI MS: m/z 600 (MH+)

¹H NMR (500 MHz, acetone-d₆) δ ppm 2.06 (m, 4H) 2.62 (d, J=1.0 Hz, 3H)3.02-3.32 (m, 2H) 3.56 (br. s., 2H) 3.63 (dd, J=11.4, 9.6 Hz, 1H) 3.91(dd, J=11.4, 2.4 Hz, 1H) 4.27 (m, 1H) 4.57 (br. s., 2H) 4.73 (dd,J=10.2, 8.2 Hz, 1H) 4.84 (m, 1H) 5.35 (m, 2H) 7.05 (dd, J=8.8, 2.3 Hz,1H) 7.16 (d, J=1.7 Hz, 1H) 7.31 (d, J=1.8 Hz, 1H) 7.35 (m, 1H) 7.38 (d,J=1.0 Hz, 1H) 7.43 (m, 2H) 7.52 (d, J=9.1 Hz, 1H) 7.57 (d, J=7.6 Hz, 2H)8.16 (br. s., 1H) 10.80 (br. s., 1H) 13.38 (br. s., 1H)

Deprotection(8R)—N-(5-{[5-({5-[(3-amino-3-oxopropyl)carbamoyl]-1-methyl-1H-pyrrol-3-yl}carbamoyl)-1-methyl-1H-pyrrol-3-yl]carbamoyl}-1-methyl-1H-pyrrol-3-yl)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6-carboxamide[(II)] (Compd. 10)

A solution of the intermediate (15 mg, 0.0182 mmol), 25% aq. HCO₂NH₄(0.15 mL) and 10% Pd—C (15 mg) in THF (3 mL) was stirred for 3 h undernitrogen atmosphere. The reaction mixture was filtered through Celiteand concentrated to yield after chromatographic purification (DCM-MeOH10:1) compound 10 (6.7 mg, 50%).

ESI MS: m/z 734 (MH+)

Analogously the following compounds have been prepared:

(8S)—N-(5-{[5-({5-[(3-amino-3-oxopropyl)carbamoyl]-1-methyl-1H-pyrrol-3-yl}carbamoyl)-1-methyl-1H-pyrrol-3-yl]carbamoyl}-1-methyl-1H-pyrrol-3-yl)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6-carboxamide[(II)] (Compd. 11)

ESI MS: m/z 734 (MH+)

N-(2-{[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1H-indol-5-yl)-5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indole-2-carboxamide[(II)] (Compd. 17)

ESI MS: m/z 668 (MH+)

¹H NMR (500 MHz, acetone-d₆) δ ppm 2.19-2.25 (m, 4H) 2.60 (s, 3H) 3.39(br. s., 2H) 3.81 (m, 2H) 3.88 (dd, J=11.1, 2.5 Hz, 1H) 3.91-3.96 (br.s,2H) 4.24 (m, 1H) 4.51 (m, 1H) 4.72 (dd, J=10.8, 7.8 Hz, 1H) 4.80-4.82(d, J=10.8, 1H) 7.01 (dd, J=8.9, 2.4 Hz, 1H) 7.22 (s, 1H) 7.26 (d, J=2.3Hz, 1H) 7.28 (s, 1H) 7.33 (s, 1H) 7.49 (d, J=8.9 Hz, 1H) 7.55 (m, 1H)7.60 (m, 1H) 7.90 (br. s., 1H) 8.29 (d, J=1.8 Hz, 1H)

[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]{5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}methanonehydrochloride [(II)] (Compd. 37)

ESI MS: m/z 510 (MH+)

¹H NMR (500 MHz, acetone-d₆) δ ppm 2.10-2.29 (m, 4H) 2.58 (d, J=1.3 Hz,3H) 3.33 (m, 2H) 3.54 (dd, J=11.4, 10.1 Hz, 1H) 3.80 (m, 2H) 3.89 (m,3H) 4.20 (m, 1H) 4.51 (m, 2H) 4.68 (dd, J=10.2, 8.2 Hz, 1H) 4.77 (m, 1H)7.04 (dd, J=8.9, 2.4 Hz, 1H) 7.12 (s, 1H) 7.29 (d, J=2.3 Hz, 1H) 7.31(d, J=0.5 Hz, 1H) 7.49 (d, J=8.9 Hz, 1H) 7.87 (br. s., 1H)

[(8R)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]{5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}methanonehydrochloride [(II)] (Compd. 38)

ESI MS: m/z 510 (MH+)

Step a

A solution of compound 10 (5 mg, 0.0068 mmol), in DMF (5 mL), wastreated with a solution of NaHCO₃ in water (3 mL, 15 mg NaHCO₃/mL). Thereaction mixture was stirred for 4 h, EtOAc was added and the resultingorganic layer was washed with brine (×4), dried (Na₂SO₄) andconcentrated in vacuo. The crude residue was purified by flashchromatography (DCM-MeOH 10:1) to afford the compound 9 (3.9 mg, 82%).

(3bS,4aR)—N-(5-{[5-({5-[(3-amino-3-oxopropyl)carbamoyl]-1-methyl-1H-pyrrol-3-yl}carbamoyl)-1-methyl-1H-pyrrol-3-yl]carbamoyl}-1-methyl-1H-pyrrol-3-yl)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indole-6(8H)-carboxamide[(I)] (Compd. 9)

ESI MS: m/z 698 (MH+)

¹H NMR (500 MHz, dichloromethane-d₂) δ ppm 1.37 (m, 1H) 2.12 (dd, J=7.6,4.6 Hz, 1H) 2.18 (d, J=0.9 Hz, 3H) 2.47 (t, J=6.6 Hz, 2H) 3.26 (dt,J=7.6, 5.0 Hz, 1H) 3.53 (t, J=6.6 Hz, 2H) 3.85 (s, 3H) 3.88 (s, 6H) 4.05(d, J=10.4 Hz, 1H) 4.14 (m, 1H) 6.64 (d, J=2.1 Hz, 1H) 6.73 (s, 1H) 6.82(m, 2H) 6.99 (d, J=1.8 Hz, 1H) 7.17 (d, J=1.8 Hz, 1H) 7.20 (d, J=1.8 Hz,1H) 7.29 (d, J=0.9 Hz, 1H)

Analogously the following compounds have been prepared:

(3bR,4aS)—N-(5-{[5-({5-[(3-amino-3-oxopropyl)carbamoyl]-1-methyl-1H-pyrrol-3-yl}carbamoyl)-1-methyl-1H-pyrrol-3-yl]carbamoyl}-1-methyl-1H-pyrrol-3-yl)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indole-6(8H)-carboxamide[(I)] (Compd. 12)

ESI MS: m/z 698 (MH+)

Example 4 Step c, Deprotection

Step c(8S)-6-({5[({5-[(tert-butoxycarbonyl)amino]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl4-methylpiperazine-1-carboxylate [(II)] (Compd. 22)

To a solution of tert-butyl{2-[(2-{[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1H-indol-5-yl)carbamoyl]-1H-indol-5-yl}carbamate,(compd. 18) (42 mg, 0.064 mmol) in dry DCM (6 mL)4-methylpiperazine-1-carbonyl chloride hydrochloride (XIII) (39 mg,0.193 mmol) and N,N-dimethylaminopyridine (27 mg, 0.212 mmol) wereadded. The reaction mixture was stirred at room temperature undernitrogen atmosphere for 16 h. The solvent was evaporated and the residuewas dissolved in EtOAc, the resulting organic layer was washed withbrine (×3), dried (Na₂SO₄) and concentrated in vacuo. The crude residuewas purified by flash chromatography (DCM-MeOH 95:5) to afford thecompd. 22 (30 mg, 59%).

ESI MS: m/z 796 (MH⁺)

¹H NMR (500 MHz, acetone-d₆) δ ppm 2.22 (s, 3H) 2.65 (d, J=0.9 Hz, 3H)2.39-2.91 (m, 4H) 3.72 (dd, J=11.6, 9.5 Hz, 1H) 3.96 (dd, J=11.6, 3.1Hz, 1H) 3.49-4.14 (m, 4H) 4.37 (m, 1H) 4.79-4.85 (m, 1H) 4.87-4.93 (m,1H) 7.27 (d, J=1.5 Hz, 1H) 7.28 (s, 1H) 7.34-7.38 (m, 1H) 7.41 (s, 1H)7.50 (d, J=8.8 Hz, 1H) 7.56 (d, J=8.5 Hz, 1H) 7.62-7.65 (m, 1H) 7.96(br. s., 1H) 8.38 (s, 1H) 9.60 (s, 1H) 10.86 (br. s., 1H) 10.90 (br. s.,1H)

By analogous procedure the following products have been prepared:

(8R)-6-({5[({5-[(tert-butoxycarbonyl)amino]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl4-methylpiperazine-1-carboxylate [(II)] (Compd. 27)

ESI MS: m/z 796 (MH⁺)

(8S)-8-(chloromethyl)-1-methyl-6-[(5-{[(5-nitro-1H-indol-2-yl)carbonyl]amino}-1H-indol-2-yl)carbonyl]-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl4-methylpiperazine-1-carboxylate [(II)] (Compd. 31)

ESI MS: m/z 726 (MH⁺)

(8R)-8-(chloromethyl)-1-methyl-6-[(5-{[(5-nitro-1H-indol-2-yl)carbonyl]amino}-1H-indol-2-yl)carbonyl]-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl4-methylpiperazine-1-carboxylate [(II)] (Compd. 32)

ESI MS: m/z 726 (MH⁺)

Deprotection(8S)-6-[(5-{[(5-amino-1H-indol-2-yl)carbonyl]amino}-1H-indol-2-yl)carbonyl]-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl4-methylpiperazine-1-carboxylate hydrochloride [(II)] (Compd. 23)

A solution of compd. 22 (22 mg, 0.0276 mmol) in 3.5 M HCl-EtOAc (5 mL)was stirred for 30 minutes before removing the solvent under a steadystream of nitrogen and affording the desired product as hydrochloridesalt (18 mg, 89%).

ESI MS: m/z 696 (MH⁺)

¹H NMR (500 MHz, DMSO-d₆) δ ppm 2.59 (s, 3H) 2.76 (br. s, 3H) 3.05-3.42(m, 4H) 3.75 (dd, J=10.8, 7.9 Hz, 1H) 3.95-4.01 (m, 1H) 3.58-4.08 (m,4H) 4.32-4.40 (m, 1H) 4.67 (d, J=11.1 Hz, 1H) 4.75-4.83 (m, 1H) 6.92(br. s., 1H) 7.21 (br. s., 1H) 7.23 (s, 1H) 7.30 (br. s., 1H) 7.37 (d,J=8.4 Hz, 1H) 7.49-7.52 (m, 1H) 7.55 (s, 1H) 7.57-7.60 (m, 1H) 8.16 (s,1H) 8.24 (s, 1H) 10.15 (br. s., 1H) 11.67 (br. s., 1H) 11.70 (s, 1H)

By analogous procedure the following products have been prepared:

(8R)-6-[(5-{[(5-amino-1H-indol-2-yl)carbonyl]amino}-1H-indol-2-yl)carbonyl]-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl4-methylpiperazine-1-carboxylate hydrochloride [(II)] (Compd. 28)

ESI MS: m/z 696 (MH⁺)

(8S)-8-(chloromethyl)-6-[(5-{[(5-hydroxy-1H-indol-2-yl)carbonyl]amino}-1H-indol-2-yl)carbonyl]-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl4-methylpiperazine-1-carboxylate [(II)] (Compd. 33)

ESI MS: m/z 697 (MH⁺)

(8R)-8-(chloromethyl)-6-[(5-{[(5-hydroxy-1H-indol-2-yl)carbonyl]amino}-1H-indol-2-yl)carbonyl]-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl4-methylpiperazine-1-carboxylate [(II)] (Compd. 34)

ESI MS: m/z 697 (MH⁺)

Example 5 Step c, Deprotection

Step c Preparation of tert-butyl(8S)-8-(chloromethyl)-6-({5-[(1H-indol-2-ylcarbonyl)amino]-1H-indol-2-yl}carbonyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-ylpiperazine-1,4-dicarboxylate[(II)]

N-(2-{[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1H-indol-5-yl)-1H-indole-2-carboxamide(111 mg, 0.2 mmol), prepared as reported in GB2344818, was dissolved indry DCM (15 mL) and to this solution tert-butyl4-(chlorocarbonyl)piperazine-1-carboxylate (100 mg, 0.4 mmol) andN,N-dimethylaminopyridine (55 mg, 0.45 mmol) were added. The reactionmixture was stirred at room temperature under nitrogen atmosphere for 16h. The solvent was evaporated and the residue was dissolved in EtOAc,the resulting organic layer was washed with brine (×4), dried (Na₂SO₄)and concentrated in vacuo. The crude residue was purified by flashchromatography (hexane-acetone 7:3) to afford the title compound (30 mg,19%).

ESI MS: m/z 767 (MH⁺)

¹H NMR (500 MHz, acetone-d₆) δ ppm 1.48 (s, 9H) 2.65 (d, J=1.01 Hz, 3H)3.55 (br. s., 4H) 3.60 (br. s., 2H) 3.73 (dd, J=11.36, 9.59 Hz, 1H) 3.79(br. s., 2H) 3.97 (dd, J=11.36, 2.78 Hz, 1H) 4.33-4.42 (m, 1H) 4.80-4.86(m, 1H) 4.89-4.94 (m, 1H) 7.10 (t, J=7.19 Hz, 1H) 7.23-7.27 (m, 1H) 7.28(s, 1H) 7.34 (d, J=1.51 Hz, 1H) 7.42 (s, 1H) 7.53-7.57 (m, 1H) 7.60 (d,J=8.08 Hz, 1H) 7.61-7.65 (m, 1H) 7.67 (d, J=8.08 Hz, 1H) 8.29 (s, 1H)8.38 (d, J=1.26 Hz, 1H) 9.56 (s, 1H) 10.89 (br. s., 2H).

Deprotection(8S)-8-(chloromethyl)-6-({5-[(1H-indol-2-ylcarbonyl)amino]-1H-indol-2-yl}carbonyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-ylpiperazine-1-carboxylate [(II)] (Compd. 24)

A solution of the intermediate (25 mg, 0.0326 mmol) in 3.5 M HCl-EtOAc(5 mL) was stirred for 2 h. After evaporation of the solvent under asteady stream of nitrogen, the residue was dried in vacuo to afford thecompd. 9 (11 mg, 48%).

ESI MS: m/z 667 (MH⁺)

Example 6 Step e′″N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N˜5˜-carbamoyl-N-[4-({[{3-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]-2,2-dimethylpropyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide[(IV)] (Compd. 35)

Step e′″

To a solution ofN-(2-{[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1H-indol-5-yl)-5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indole-2-carboxamide(Compd 17) (5 mg, 0.0071 mmol) in DCM (1 ml) and THF (0.5 mL) were added4-nitrophenyl chloroformate (3.1 mg, 0.0156 mmol) and triethylamine (2.2mL, 0.0156 mmol). The resulting mixture was stirred at room temperaturefor 6 hours.N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-{4-[({[2,2-dimethyl-3-(methylamino)propyl](methyl)carbamoyl}oxy)methyl]phenyl}-L-ornithinamide (XX)′ (16 mg, 0.018mmol) and triethylamine (3 mL, 0.021 mmol) were added. The mixture thusobtained was stirred at room temperature overnight, diluted with DCM andwashed with a saturated solution of aqueous NaHCO₃. The crude residuewas purified by flash chromatography (DCM-MeOH 100:15) to afford thetitle compound (2 mg, 20%).

ESI MS: m/z 1422 (MH+)

¹H NMR (500 MHz, methanol-d₃) δ ppm 0.82-1.45 (m 29H) 2.68 (s, 3H)2.90-3.61 (m, 20H) 3.84-4.34 (m, 8H) 4.49 (m, 1H) 5.04-5.14 (m, 2H)6.70-6.77 (m, 2H) 7.01 (dd, J=9.0, 2.4, 1H) 7.18-7.71 (m, 11H) 8.11 (br.s., 2H)

Analogously the following compounds have been prepared:

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N˜5˜-carbamoyl-N-[4-({[{3-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]-2,2-dimethylpropyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide[(IV)] (Compd 36)

ESI MS: m/z 1422 (MH+)

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-[4-({[{3-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]-2,2-dimethylpropyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide[(IV)] (Compd 39)

ESI MS: m/z 1264 (MH+)

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-[4-({[{3-[({[(8R)-8-(chloromethyl)-1-methyl-6-({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl]carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]-2,2-dimethylpropyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide[(IV)](Compd 40)

ESI MS: m/z 1264 (MH+)

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-[4-({[(3-{[({(8S)-8-(chloromethyl)-1-methyl-6-[(2E)-3-{5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}prop-2-enoyl]-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl}oxy)carbonyl](methyl)amino}-2,2-dimethylpropyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide[(IV)] (Compd 41)

ESI MS: m/z 1290 (MH+)

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-[4-({[(3-{[({(8R)-8-(chloromethyl)-1-methyl-6-[(2E)-3-{5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}prop-2-enoyl]-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl}oxy)carbonyl](methyl)amino}-2,2-dimethylpropyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide[(IV)] (Compd 42).

ESI MS: m/z 1290 (MH+)

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-phenylalanyl-L-leucyl-N-[4-({[{3-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]-2,2-dimethylpropyl}(methyl)carbamoyl]oxy}methyl)phenyl]glycinamide[(IV)] (Compd 46)

ESI MS: m/z 1483 (MH+)

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-phenylalanyl-L-leucyl-N-[4-({[{3-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]-2,2-dimethylpropyl}(methyl)carbamoyl]oxy}methyl)phenyl]glycinamide[(IV)] (Compd 47)

ESI MS: m/z 1325 (MH+)

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-phenylalanyl-L-leucyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]glycinamide[(IV)] (Compd 48)

ESI MS: m/z 1441 (MH+)

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-phenylalanyl-L-leucyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]glycinamide[(IV)] (Compd 49)

ESI MS: m/z 1283 (MH+)

Example 7 Step c, Deprotection, Step g

Step cN-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-valyl-N⁵-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide[(II)]

To a solution ofN-(2-{[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1H-indol-5-yl)-5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indole-2-carboxamidehydrochloride (13 mg, 0.02 mmol) in THF/DCM 1:1 (2 mL) at 0° C. undernitrogen atmosphere, were added 4-nitrophenyl chloroformate (20 mg, 0.1mmol) and triethylamine (20 mL, 0.14 mmol). The mixture was stirred atroom temperature for 6 hours, solvent was evaporated and the residue wasisolated by filtration after treatment with diethyether. The resultingsolid compound was diluted with 1 mL of DCM (10% DMF) andN-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-valyl-N⁵-carbamoyl-N-{4-[({methyl[2-(methylamino)ethyl]carbamoyl}oxy)methyl]phenyl}-L-ornithinamidehydrochloride (38 mg, 0.05 mmol) and trietylamine (7 mL, 0.05 mmol) wereadded. The mixture was stirred overnight, solvents were evaporated andtitle compound (12 mg, 42% yield) was isolated by column chromatographypurification (DCM/MeOH 8:2).

ESI MS: m/z 1409 (MH+)

¹H NMR (500 MHz, methanol-d₄) δ ppm 2.07 (br. s., 4H) 2.58 (s, 3H)2.93-3.09 (m, 6H) 3.40-3.69 (m, 8H) 4.11-4.40 (m, 3H) 4.43-4.81 (m, 3H)4.99-5.17 (m, 2H) 6.92-7.44 (m, 14H) 7.46-7.64 (m, 4H) 7.75 (br. s., 3H)8.01-8.22 (m, 2H) 8.01-8.06 (m, 1H)

Analogously the following compound has been prepared:

N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-valyl-N⁵-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide

ESI MS: m/z 1251 (MH+)

¹H NMR (500 MHz, methanol-d₄) δ ppm 0.94-1.01 (m, 6H) 1.51-1.95 (m, 4H)2.12 (br. s., 5H) 2.48-2.62 (m, 3H) 2.89-3.05 (m, 4H) 3.40-3.54 (m, 4H)3.46-3.65 (m, 4H) 3.58-3.74 (m, 2H) 3.75-4.05 (m, 1H) 4.08-4.42 (m, 3H)4.50 (m, 1H) 4.55-4.81 (m, 4H) 4.96-5.20 (m, 2H) 6.92-7.81 (m, 17H) 8.04(br. s., 1H)

DeprotectionL-valyl-N⁵-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide[(II)] (Compd 50)

To a solution ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-valyl-N⁵-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl}ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide(11 mg, 0.0078 mmol) in DMF (1 mL) was added piperidine (4 mL, 0.039mmol). The mixture was stirred 1 hour at room temperature, solvent wasevaporated and the resulting title compound was used without furtherpurification. Analogously the following compound has been prepared:

L-valyl-N⁵-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide[(II)] (Compd 51)

Step gN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide[(IV)] (Compd 52)

To a solution ofL-valyl-N⁵-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide(4.6 mg, 0.0039 mmol) in 1 mL of DCM (10% DMF), were added1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione(3.6 mg, 0.0117 mmol) and triethylamine (2.2 mL, 0.0156 mmol). Themixture was stirred at room temperature for 3 hours, solvents wereevaporated and the title compound (2.2 mg, 41% yield) was isolated bycolumn chromatography purification (DCM/MeOH 8:2).

ESI MS: m/z 1380 (MH+)

¹H NMR (500 MHz, methanol-d₄) δ ppm 1.98 (br. s., 4H) 2.60-2.63 (m, 3H)2.89-3.11 (m, 6H) 3.39-3.72 (m, 10H) 4.07-4.75 (m, 7H) 4.98-5.17 (m, 2H)6.74 (br. s., 2H) 7.00 (m, 2H) 7.07-7.45 (m, 8H) 7.52 (br. s., 3H)8.07-8.11 (m, 1H)

Analogously the following compounds have been prepared:

N-[19-(2,5-dioxo-2,5-d ihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecan-1-oyl]-L-valyl-N⁵-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide[(IV)] (Compd 53)

ESI MS: m/z 1585 (MH+)

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide[(IV)] (Compd 54)

ESI MS: m/z 1222 (MH+)

¹H NMR (400 MHz, dmf-d7) δ ppm 0.95 (t, J=7.8 Hz, 6H) 2.16 (m, 1H) 2.66(br. s., 3H) 2.99-3.09 (m, 3H) 3.09-3.29 (m, 3H) 3.59 (br. s., 2H) 3.69(br. s., 2H) 3.81 (br. s., 2H) 4.04 (d, J=10.7 Hz, 1H) 4.31-4.47 (m, 2H)4.61 (br. s., 1H) 4.83 (br. s., 2H) 5.11 (d, J=15.7 Hz, 2H) 5.60 (s, 2H)6.29 (br. s., 1H) 7.00 (m, 3H) 7.30 (m, 2H) 7.53 (d, J=8.5 Hz, 2H) 7.88(d, J=8.2 Hz, 1H) 8.13 (d, J=7.8 Hz, 1H) 8.27 (s, 1H) 10.1 (m, 1H) 11.61(br.s., 1H)

Preparation of the IntermediateN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-{4-[({[2,2-dimethyl-3-(methylamino)propyl](methyl)carbamoyl}oxy)methyl]phenyl}-L-ornithinamide(XX)′

Step 1 N,N′-(2,2-dimethylpropane-1,3-diyl)diformamide

In a round bottomed flask, commercially available2,2-dimethylpropane-1,3-diamine (851 mg, 8.32 mmol) was reacted withethylformate (4.2 ml). The reaction mixture was stirred at 60° C. for 3hours, until no starting material was detectable (TLC analysis,MeOH:CH₂Cl₂=2:8). The reaction mixture was then evaporated under vacuum,affording the crude product (1.4 g, oil).

ESI MS: m/z 159 (MH+)

¹H NMR (600 MHz, DMSO-d₆) ppm 0.79 (s, 6H) 2.92 (d, J=6.41 Hz, 4H) 7.94(br. s., 2H) 8.05-8.09 (m, 2H)

Step 2 N,N′,2,2-tetramethylpropane-1,3-diamine

In a dried round bottomed flask, containingN,N′-(2,2-dimethylpropane-1,3-diyl)diformamide (1.4 g, 8.85 mmol) cooledat 0° C. under argon atmosphere, a solution (24 ml) of Lithium AluminiumHydride in tetrahydrofuran (1M) was added. The reaction mixture wasstirred at room temperature for 28 hours. After cooling at 0° C., asolution of water in tetrahydrofuran was added, and the resultingmixture was filtered on a celite pad. The filtrate was dried overanhydrous sodium sulfate, filtered and the product thus obtained as atetrahydrofuran solution was used without further purification in thenext step.

Step 32-(biphenyl-4-yl)propan-2-yl[2,2-dimethyl-3-(methylamino)propyl]methylcarbamate

Commercial methyl4-[({[2-(biphenyl-4-yl)propan-2-yl]oxy}carbonyl)oxy]benzoate (5.54 mmol,2.16 g) was added to the tetrahydrofuran solution ofN,N′,2,2-tetramethylpropane-1,3-diamine. The reaction mixture wasstirred at room temperature for 24 hours. The solvent was thenevaporated under vacuum and the residue was purified by flashchromatography (MeOH:CH₂Cl₂=1:9) affording the desired product (848 mg).

MS (ESI): 369 (MH+)

¹H NMR (600 MHz, DMSO-d₆) δ ppm 0.73-0.83 (m, 5H) 0.91 (br. s., 3H)1.65-1.77 (m, 6H) 2.78 (br. s., 1H) 7.32-7.38 (m, 1H) 7.41 (d, J=8.43Hz, 2H) 7.46 (t, J=7.69 Hz, 2H) 7.58-7.69 (m, 4H)

Analogously the following compound has been prepared:

2-(biphenyl-4-yl)propan-2-yl methyl[2-(methylamino)ethyl]carbamate

MS (ESI): 327 (MH+)

¹H NMR (401 MHz, DMSO-d₆) δ ppm 1.70-1.75 (m, 6H) 2.23-2.39 (m, 3H)7.30-7.39 (m, 1H) 7.40-7.49 (m, 4H) 7.58-7.63 (m, 2H) 7.63-7.68 (m, 2H)

Step 4N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[11-(biphenyl-4-yl)-4,6,6,8,11-pentamethyl-3,9-dioxo-2,10-dioxa-4,8-diazadodec-1-yl]phenyl)-N⁵-carbamoyl-L-ornithinamide

To a solution ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N5-carbamoyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-ornithinamide(0.492 mmol, 363 mg) in anhydrous dimethylsulfoxide (1.0 ml), a solutionof2-(biphenyl-4-yl)propan-2-yl[2,2-dimethyl-3-(methylamino)propyl]methylcarbamate(0.393 mmol, 145 mg) in anhydrous dimethylsulfoxide (0.9 ml) andtriethylamine (1.5 mmol, 150 mg) were added. The reaction mixture wasstirred at room temperature until no starting material was detectable,then treated with n-hexane (3×6 ml), and the crude residue was usedwithout further purification in the next step.

MS (ESI): 967 (MH+).

Analogously the following compound has been prepared

N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-valyl-N-{4-[10-(biphenyl-4-yl)-4,7,10-trimethyl-3,8-dioxo-2,9-dioxa-4,7-diazaundec-1-yl]phenyl}-N⁵-carbamoyl-L-ornithinamide

MS (ESI): 954 (MH+)

¹H NMR (500 MHz, DMSO-d₆) δ ppm 0.86 (dd, J=13.34, 6.79 Hz, 6H) 1.69(br. s., 6H) 1.86-2.05 (m, 1H) 2.76 (m, J=9.30 Hz, 2H) 2.92 (d, J=14.95Hz, 3H) 3.01 (m, J=6.10 Hz, 1H) 3.15-3.15 (m, 0H) 3.38-3.53 (m, 2H) 3.93(t, J=7.63 Hz, 1H) 4.18-4.26 (m, 2H) 4.26-4.34 (m, 1H) 4.42 (br. s., 1H)4.92-5.06 (m, 2H) 5.39 (s, 2H) 5.96 (t, J=6.02 Hz, 1H) 7.18-7.48 (m,12H) 7.52-7.66 (m, 6H) 7.70-7.77 (m, 2H) 7.88 (d, J=7.47 Hz, 2H) 8.11(br. s., 1H) 10.06 (br. s., 1H)

Step 5 The Title Intermediate

The crude productN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]L-valyl-N-{4-[11-(biphenyl-4-yl)-4,6,6,8,11-pentamethyl-3,9-dioxo-2,10-dioxa-4,8-diazadodec-1-yl]phenyl}-N⁵-carbamoyl-L-ornithinamidewas treated with dichloroacetic acid (7.8 mmol). The reaction mixturewas stirred at room temperature, until no starting material wasdetectable, then purified by flash column chromatography(EtOH:CH₂Cl₂=1.5:8.5) on silica gel, affording the desired product (44.0mg, white solid).

MS (ESI): 729 (MH+)

¹H NMR (500 MHz, METHANOL-d₄) δ ppm 0.97 (d, J=4.42 Hz, 3H) 0.98 (d,J=4.27 Hz, 3H) 1.04 (s, 6H) 1.23-1.35 (m, 4H) 1.50-1.68 (m, 7H) 1.76(dtd, J=13.90, 9.26, 9.26, 5.11 Hz, 1H) 1.86-1.95 (m, 1H) 2.07 (dq,J=13.90, 6.86 Hz, 1H) 2.26-2.31 (m, 2H) 2.57 (s, 3H) 2.67 (s, 2H) 3.04(s, 3H) 3.11 (dt, J=13.50, 6.67 Hz, 1H) 3.16-3.22 (m, 1H) 3.23 (br. s.,2H) 3.48 (t, J=7.09 Hz, 2H) 4.14 (d, J=7.47 Hz, 1H) 4.49 (dd, J=9.07,5.11 Hz, 1H) 5.13 (s, 2H) 5.94 (s, 3H) 6.75-6.82 (m, 1H) 7.36 (d, J=8.54Hz, 2H) 7.60 (d, J=8.69 Hz, 2H).

Preparation of IntermediateN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-{4-[({methyl[2-(methylamino)ethyl]carbamoyl}oxy)methyl]phenyl}-L-ornithinamide

Step 6N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[11-(biphenyl-4-yl)-4,6,6,8,11-pentamethyl-3,9-dioxo-2,10-dioxa-4,8-diazadodec-1-yl]phenyl}-N⁵-carbamoyl-L-ornithinamide

To a solution of the productN-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-valyl-N-{4-[10-(biphenyl-4-yl)-4,7,10-trimethyl-3,8-dioxo-2,9-dioxa-4,7-diazaundec-1-yl]phenyl}-N⁵-carbamoyl-L-ornithinamide[prepared as reported above under step 4] (0.063 mmol, 60 mg) inanhydrous dimethylformamide (0.8 ml), piperidine (0.32 mmol, 27.2 mg)was added. The reaction mixture was stirred at room temperature until nostarting material was detectable. The solvent was evaporated undervacuum and the crude thus obtained was used without further purificationin the next step.

MS (ESI): 732 (MH+)

Step 7N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[10-(biphenyl-4-yl)-4,7,10-trimethyl-3,8-dioxo-2,9-dioxa-4,7-diazaundec-1-yl]phenyl}-N⁵-carbamoyl-L-ornithinamide

To a solution of the crude productN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]L-valyl-N-{4-[11-(biphenyl-4-yl)-4,6,6,8,11-pentamethyl-3,9-dioxo-2,10-dioxa-4,8-diazadodec-1-yl]phenyl}-N⁵-carbamoyl-L-ornithinamide(0.0629 mmol) in anhydrous dimethylformamide (1.5 ml),1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione(0.189 mmol, 58.3 mg) and triethylamine (0.252 mmol, 25.5 mg) wereadded. The reaction mixture was stirred at room temperature until nostarting material was detectable. The solvent was evaporated undervacuum and the crude was purified by flash column chromatography(eluant:EtOH:CH₂Cl₂=1:9) on silica gel (230-400 mesh), affording thedesired product (34 mg, white wax)

MS (ESI): 925 (MH+)

¹H NMR (500 MHz, DMSO-d₆) δ ppm 0.83 (dd, J=15.79, 6.79 Hz, 6H)1.31-1.40 (m, 1H) 1.60 (br. s., 1H) 1.69 (s, 6H) 1.90-2.02 (m, 1H)2.05-2.23 (m, 2H) 2.89-2.96 (m, 3H) 3.01 (br. s., 1H) 3.34-3.38 (m, 2H)3.46 (m, J=8.24 Hz, 2H) 4.19 (t, J=7.40 Hz, 1H) 4.38 (br. s., 1H)4.95-5.05 (m, 2H) 5.40 (s, 2H) 5.97 (d, J=12.20 Hz, 1H) 6.98-7.02 (m,2H) 7.30 (d, J=8.85 Hz, 2H) 7.37 (dd, J=15.40, 7.93 Hz, 3H) 7.45 (m,J=7.17 Hz, 2H) 7.52-7.67 (m, 6H) 7.80 (d, J=8.24 Hz, 1H) 8.08 (d, J=6.71Hz, 1H) 9.76-10.18 (m, 1H)

Step 8 The Title Intermediate

Reacting the intermediate prepared under step 6 under reactionconditions reported above in step 5, the title compound as a whitepowder was obtained

MS (ESI): 687 (MH+)

1. A compound of formula (I) or (II)

wherein R1 and R2 taken together form a group (D) or (G):

wherein R5 is hydrogen, linear or branched C₁-C₄ alkyl, linear or branched C₁-C₄ hydroxyalkyl or linear or branched C₁-C₄ aminoalkyl; R3 and R4 are, each independently, hydrogen or a group selected from an optionally substituted linear or branched C₁-C₄ alkyl and linear or branched C₁-C₄ hydroxyalkyl; R6 is a leaving group; T is null or N; BM is a DNA binding moiety of formula (V):

wherein: X is null, linear or branched C₁-C₄ alkyl, linear or branched C₂-C₄ alkenyl or linear or branched C₂-C₄ alkynyl; Y and Y′ are independently an optionally substituted aryl or heteroaryl; U is a moiety of formula (VI) or (VII):

wherein R3 is as defined above; q is an integer from 0 to 4; L1 is hydrogen or L, wherein L is null or a conditionally-cleavable moiety; W1 is null or a self-immolative system comprising one or more self-immolative groups; Z1 is null or a peptidic, non peptidic or hydrid peptidic and non peptidic linker; provided that a compound of formula (I) or a compound of formula (II) wherein L1 is hydrogen is excluded when 1) both T and X are null, q is 0 and Y′ is an heterocyclyl moiety of formula (VIII), (VIII)′ or (VIII)″:

or 2) both T and X are null, q is 1, U is a group of formula (VII), Y is an heterocyclyl moiety of formula (IX)

and Y′ is an heterocyclyl moiety of formula (VIII)′″

wherein Q is N or O, and R21 is hydrogen or a group selected from —N(C₂H₅)₂ and C(NH)NH₂; and the pharmaceutically acceptable salts thereof.
 2. A compound of formula (I) or (II) according to claim 1, wherein R1 and R2 taken together form a group (D)

wherein R5 is linear or branched C₁-C₄ alkyl.
 3. A compound of formula (II) according to claim 2, wherein R6 is halide and L1 is hydrogen or a conditionally-cleavable moiety of formula (Xj)

wherein R9 is hydrogen, hydroxy or an optionally substituted group selected from linear or branched C₁-C₄ alkyl, linear or branched C₁-C₄ hydroxyalkyl, linear or branched C₁-C₄ sulfhydrylalkyl and linear or branched C₁-C₄ aminoalkyl.
 4. A compound of formula (III) or (IV)

wherein BM is a DNA binding moiety of formula (V)′:

wherein: X, Y, U, Y′ and q are as defined above; A is an atom selected from —O—, —NH—, —CO—; A′ is null or A, wherein A is as defined above; L is null or a conditionally-cleavable moiety; W is null or a self-immolative system comprising one or more self-immolative groups; Z is null or a peptidic, non peptidic or hybrid peptidic and non peptidic linker; RM is null or a reactive moiety attached to one or more of A, L, W or Z groups; RM1 is null or a reactive moiety attached to one or more of L1, W1 or Z1 groups; and R1, R2, R3, R4, R6, T, BM, L1, W1 and Z1 are as defined in claim 1; provided that 1) a compound of formula (IV) is excluded when A1 is null and RM1 is null; 2) a compound of formula (III) or (IV) is excluded when a) both T and X are null, q is 0 and Y′ is an heterocyclyl moiety of formula (VIII)^(IV)

wherein Q is —O—, —S—, —NR14, wherein R14 is hydrogen, C₁-C₄ alkyl or C₁-C₄ hydroxyalkyl; Q1 is —CH═ or —N═; R7 and R8 are independently hydrogen, halogen, hydroxy, C₁-C₄ alkoxy, cyano, —NCOOR3, —C(NH)—NH₂ or —NR3R4, wherein R3 and R4 are as defined in claim 1; or b) both T and X are null, q is 1 or 2, U is a group of formula (VII), Y is a heterocyclyl moiety of formula (IX)′:

and Y′ is a heterocyclyl moiety of formula (VIII)^(IV)

wherein Q, Q1, R7 and R8 are as defined above; and the pharmaceutically acceptable salts thereof.
 5. A compound of formula (III) or (IV) according to claim 4, wherein R1 and R2 taken together form a group (D)

wherein R5 is linear or branched C₁-C₄ alkyl.
 6. A compound of formula (IV) according to claim 5, wherein R6 is halide; and A′ is null and L1 is L, wherein L is null or a conditionally-cleavable moiety.
 7. A compound of formula (III) or (IV) according to claim 5 or 6, wherein L and L1 are independently null or a conditionally-cleavable moiety selected from NHCO—R9 (Xa); —NHCONH—R9 (Xb); —NHCOO—R9 (Xc); —NH—R9 (Xd);

wherein: R9 and R10 are, each independently, null, hydrogen, hydroxy or an optionally substituted group selected from linear or branched C₁-C₄ alkyl, linear or branched C₁-C₄ hydroxyalkyl, linear or branched C₁-C₄ sulfhydrylalkyl and linear or branched C₁-C₄ aminoalkyl; n is an integer from 0 to 2 and n1 is an integer from 0 to
 4. 8. A compound of formula (III) or (IV) according to claim 5, 6 or 7, wherein W and W1 are independently a self-immolative system comprising one or more self-immolative groups independently selected from:

wherein one of R9 and R10 is null and the other is as defined in claim 7; R11 and R12 are, each independently, hydrogen, halogen, methyl, ethyl or linear or branched C₁-C₄ hydroxymethyl; m is an integer from 0 to 3; and A₁ is CH₂, CH₂N—R12 or N—R12, wherein R12 is as defined above.
 9. A compound of formula (III) or (IV) according to claim 5, 6, 7 or 8, wherein Z and Z1 are independently a peptidic linker, preferably a dipeptide or a tripeptide linker, or is a non peptidic linker containing an oligoethylene glycol or polyethylene glycol moiety or a derivative thereof selected from:

wherein one of R9 and R10 is null and the other is are as defined in claim 7; and p is an integer from 1 to
 20. 10. A compound of formula (III) or (IV) according to claim 5, 6, 7, 8 or 9, wherein RM and RM1 are independently a reactive moiety selected from

wherein R13 is C₁-C₃ alkyl or an electron-withdrawing group comprising NO₂ and CN group; r is an integer from 0 to 7; and R11 and R12 are as defined in claim
 8. 11. A compound according to claims 1 or 4, or a pharmaceutically acceptable salt thereof, which is selected from the group consisting of: N-(6-{[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1H-indol-3-yl)-1H-indole-6-carboxamide, N-(5-{[(8R)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1-methyl-1H-pyrrol-3-yl)-1-methyl-4-{[(1-methyl-4-nitro-1H-pyrrol-2-yl)carbonyl]amino}-1H-pyrrole-2-carboxamide, (2E)-1-[(8R)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)prop-2-en-1-one, (2E)-1-[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)prop-2-en-1-one, N-(6-{[(3bR,4aS)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-3-yl)-1H-indole-6-carboxamide, 1-methyl-N-(1-methyl-5-{[(3bS,4aR)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-pyrrol-3-yl)-4-{[(1-methyl-4-nitro-1H-pyrrol-2-yl)carbonyl]amino}-1H-pyrrole-2-carboxamide, (3bS,4aR)-3-methyl-6-[(2E)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)prop-2-enoyl]-4,4-a,5,6-tetrahydro-8H-cyclopropa[c]thieno[3,2-e]indol-8-one, (3bR,4aS)-3-methyl-6-[(2E)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)prop-2-enoyl]-4,4-a,5,6-tetrahydro-8H-cyclopropa[c]thieno[3,2-e]indol-8-one, (3bS,4aR)—N-(5-{[5-({5-[(3-amino-3-oxopropyl)carbamoyl]-1-methyl-1H-pyrrol-3-yl}carbamoyl)-1-methyl-1H-pyrrol-3-yl]carbamoyl}-1-methyl-1H-pyrrol-3-yl)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indole-6(8H)-carboxamide, (8R)—N-(5-{[5-({5-[(3-amino-3-oxopropyl)carbamoyl]-1-methyl-1H-pyrrol-3-yl}carbamoyl)-1-methyl-1H-pyrrol-3-yl]carbamoyl}-1-methyl-1H-pyrrol-3-yl)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6-carboxamide, (8S)—N-(5-{[5-({5-[(3-amino-3-oxopropyl)carbamoyl]-1-methyl-1H-pyrrol-3-yl}carbamoyl)-1-methyl-1H-pyrrol-3-yl]carbamoyl}-1-methyl-1H-pyrrol-3-yl)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6-carboxamide, (3bR,4aS)—N-(5-{[5-({5-[(3-amino-3-oxopropyl)carbamoyl]-1-methyl-1H-pyrrol-3-yl}carbamoyl)-1-methyl-1H-pyrrol-3-yl]carbamoyl}-1-methyl-1H-pyrrol-3-yl)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indole-6(8H)-carboxamide, N-(3-{(1E)-3-[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]-3-oxoprop-1-en-1-yl}-1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-indole-2-carboxamide, N-(3-{(1E)-3-[(8R)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]-3-oxoprop-1-en-1-yl}-1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-indole-2-carboxamide, N-(3-{(1E)-3-[(3bR,4aS)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]-3-oxoprop-1-en-1-yl}-1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-indole-2-carboxamide, N-(3-{(1E)-3-[(3bR,4aS)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]-3-oxoprop-1-en-1-yl}-1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-indole-2-carboxamide, N-(2-{[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1H-indol-5-yl)-5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indole-2-carboxamide, tert-butyl {2-[(2-{[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1H-indol-5-yl)carbamoyl]-1H-indol-5-yl}carbamate, 5-amino-N-(2-{[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-5a,7,8,8a-tetrahydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1H-indol-5-yl)-1H-indole-2-carboxamide, tert-butyl{2-[(2-{[3bR,4aS)-3-methyl-8-oxo-4a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-5-yl)carbamoyl]-1H-indol-5-yl}carbamate, 5-amino-N-(2-{[(3bR,4aS)-3-methyl-8-oxo-4a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-5-yl)-1H-indole-2-carboxamide, (8S)-6-({5-[({5-[(tert-butoxycarbonyl)amino]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indo-4-yl 4-methylpiperazine-1-carboxylate, (8S)-6-[(5-{[(5-amino-1H-indol-2-yl)carbonyl]amino}-1H-indol-2-yl)carbonyl]-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl 4-methylpiperazine-1-carboxylate hydrochloride, (8S)-8-(chloromethyl)-6-({5-[(1H-indol-2-ylcarbonyl)amino]-1H-indol-2-yl}carbonyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl piperazine-1-carboxylate, tert-butyl{2-[(2-{[(3bS,4aR)-methyl-8-oxo-4a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-5-yl)carbamoyl]-1H-indol-5-yl}carbamate, 5-amino-N-(2-{[(3bS,4aR)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-5-yl)-1H-indole-2-carboxamide, (8R)-6-({5-[({5-[(tert-butoxycarbonyl)amino]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl 4-methylpiperazine-1-carboxylate, (8R)-6-[(5-{[(5-amino-1H-indol-2-yl)carbonyl]amino}-1H-indol-2-yl)carbonyl]-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl 4-methylpiperazine-1-carboxylate hydrochloride, N-(2-{[(3bR,4aS)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-5-yl)-5-nitro-1H-indole-2-carboxamide, N-(2-{[(3bS,4aR)-3-methyl-8-oxo-4-a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-5-yl)-5-nitro-1H-indole-2-carboxamide, (8S)-8-(chloromethyl)-1-methyl-6-[(5-{[(5-nitro-1H-indol-2-yl)carbonyl]amino}-1H-indol-2-yl)carbonyl]-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl 4-methylpiperazine-1-carboxylate, (8R)-8-(chloromethyl)-1-methyl-6-[(5-{[(5-nitro-1H-indol-2-yl)carbonyl]amino}-1H-indol-2-yl)carbonyl]-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl 4-methylpiperazine-1-carboxylate, (8S)-8-(chloromethyl)-6-[(5-{[(5-hydroxy-1H-indol-2-yl)carbonyl]amino}-1H-indol-2-yl)carbonyl]-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl 4-methylpiperazine-1-carboxylate, (8R)-8-(chloromethyl)-6-[(5-{[(5-hydroxy-1H-indol-2-yl)carbonyl]amino}-1H-indol-2-yl)carbonyl]-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl 4-methylpiperazine-1-carboxylate, N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N˜5˜-carbamoyl-N-[4-({[{3-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy}-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]-2,2-dimethylpropyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide, N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N˜5˜-carbamoyl-N-[4-({[{3-[({[(8R)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]-2,2-dimethylpropyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide, [(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]{5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}methanone, [(8R)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]{5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}methanone, N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-[4-({[{3-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]-2,2-dimethylpropyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide, N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-[4-({[{3-[({[(8R)-8-(chloromethyl)-1-methyl-6-({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]-2,2-dimethylpropyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide, N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-[4-({[(3-{[({(8S)-8-(chloromethyl)-1-methyl-6-[(2E)-3-{5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}prop-2-enoyl]-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl}oxy)carbonyl](methyl)amino}-2,2-dimethylpropyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide, N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-[4-({[(3-{[({(8R)-8-(chloromethyl)-1-methyl-6-[(2E)-3-{5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}prop-2-enoyl]-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl}oxy)carbonyl](methyl)amino}-2,2-dimethylpropyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide, (2E)-1-[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]-3-(1H-indol-3-yl)prop-2-en-1-one, N-(2-{[(8R)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1-methyl-1H-indol-5-yl)-[1-methyl-1H-indole-2-carboxamide, 1-methyl-N-(1-methyl-2-{[(3bS,4aR)-3-methyl-8-oxo-4a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-5-yl)-1H-indole-2-carboxamide, N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-phenylalanyl-L-leucyl-N-[4-({[{3-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]-2,2-dimethylpropyl}(methyl)carbamoyl]oxy}methyl)phenyl]glycinamide, N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-phenylalanyl-L-leucyl-N-[4-({[{3-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]-2,2-dimethylpropyl}(methyl)carbamoyl]oxy}methyl)phenyl]glycinamide, N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-phenylalanyl-L-leucyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]glycinamide, N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-phenylalanyl-L-leucyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]glycinamide, L-valyl-N⁵-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide, L-valyl-N⁵-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide, N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl-L-ornithinamide, N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecan-1-oyl]-L-valyl-N⁵-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide and N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide.
 12. A pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) or (II) as defined in claim 1, or a compound of formula (III) or (IV), as defined in claim 4, and at least one pharmaceutically acceptable excipient, carrier or diluent.
 13. A pharmaceutical composition according to claim 12 further comprising one or more chemotherapeutic agents.
 14. A product comprising a compound of formula (I) or (II) as defined in claim 1, or a compound of formula (III) or (IV) as defined in claim 4, or a pharmaceutically acceptable salt thereof, and one or more chemotherapeutic agents, as a combined preparation for simultaneous, separate or sequential use in anticancer therapy. 15-17. (canceled)
 18. A method for treating cancer, which comprises administering to a mammal in need thereof an effective amount of a compound of formula (I) or (II) as defined in claim 1, or a compound of formula (III) or (IV) as defined in claim
 4. 19. The method of claim 18 wherein the mammal in need thereof is a human. 20-21. (canceled) 